Abstract Background Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and ...sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder. Objectives The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). Methods This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs. Results BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+ ) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. Conclusions Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.
BACKGROUND:The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic “healthy” ...individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity.
METHODS:To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a “healthy” asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be “likely pathogenic.” Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines(1) corrected “healthy” control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3; and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs).
RESULTS:The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments.
CONCLUSIONS:Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.
Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated ...with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-β (PDGFRB) as a potential therapeutic target.
Objectives The goal of this study was to establish a patient-specific human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic polymorphic ventricular tachycardia (CPVT). Background ...CPVT is a familial arrhythmogenic syndrome characterized by abnormal calcium (Ca2+ ) handling, ventricular arrhythmias, and sudden cardiac death. Methods Dermal fibroblasts were obtained from a CPVT patient due to the M4109R heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs. The patient-specific hiPSCs were coaxed to differentiate into the cardiac lineage and compared with healthy control hiPSCs-derived cardiomyocytes (hiPSCs-CMs). Results Intracellular electrophysiological recordings demonstrated the development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by isoproterenol (1 μM) or forskolin (5 μM) increased the frequency and magnitude of afterdepolarizations and also led to development of triggered activity in the CPVT-hiPSCs-CMs. In contrast, flecainide (10 μM) and thapsigargin (10 μM) eliminated all afterdepolarizations in these cells. The latter finding suggests an important role for internal Ca2+ stores in the pathogenesis of delayed afterdepolarizations. Laser-confocal Ca2+ imaging revealed significant whole-cell Ca2+ transient irregularities (frequent local and large-storage Ca2+ -release events, broad and double-humped transients, and triggered activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation and Ca2+ overload and improved with beta-blockers. Store-overload–induced Ca2+ release was also identified in the hiPSCs-CMs and the threshold for such events was significantly reduced in the CPVT cells. Conclusions This study highlights the potential of hiPSCs for studying inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care, and aid in the development of new therapies.
RATIONALE:Targeted genetic engineering using programmable nucleases such as transcription activator–like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification ...in the human genome.
OBJECTIVE:The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.
METHODS AND RESULTS:By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN–mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt–Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.
CONCLUSIONS:Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.
The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for ...cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca(2+)-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).
RT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+)-handling proteins. Detailed laser confocal Ca(2+) imaging demonstrated spontaneous whole-cell Ca(2+)(i) transients. These transients required Ca(2+) influx via L-type Ca(2+) channels, as demonstrated by their elimination in the absence of extracellular Ca(2+) or by administration of the L-type Ca(2+) channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca(2+) store, contributing to Ca(2+)(i) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing Ca(2+)(i)). Similarly, the importance of Ca(2+) reuptake into the SR via the SR Ca(2+) ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to Ca(2+)(i) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell Ca(2+)(i) transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phospholipase C inhibitor U73122.
Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+) store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell Ca(2+)(i) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these ...unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.-- Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.
Pro-arrhythmia (development of cardiac arrhythmias as a pharmacological side effect) has become the single most common cause of the withdrawal or restrictions of previously marketed drugs. The ...development of new medications, free from these side effects, is hampered by the lack of an in vitro assay for human cardiac tissue. We hypothesized that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) assessed with a combination of single cell electrophysiology and microelectrode array (MEA) mapping can serve as a novel model for electrophysiological drug screening. Current-clamp studies revealed that E-4031 and Sotalol (IKr blockers) significantly increased hESC-CM's action potential duration and also induced after-depolarizations (the in vitro correlates of increased arrhythmogenic potential). Multicellular aggregates of hESC-CMs were then analyzed with the MEA technique. Application of class I (Quinidine, Procaineamide) and class III (Sotalol) antiarrhythmic agents, E-4031, and Cisapride (a noncardiogenic agent known to lengthen QT) resulted in dose-dependent prolongation of the corrected field potential duration (cFPD). We next utilized the MEA technique to also assess pharmacological effects on conduction. Activation maps demonstrated significant conduction slowing following administration of Na channel blockers (Quinidine and Propafenone) and of the gap junction blocker (1-heptanol). While most attention has been focused on the prospects of using hESC-derived cardiomyocytes for regenerative medicine, this study highlights the possible utilization of these unique cells also for cardiac electrophysiological studies, drug screening, and target validation.
The objective of the current study was to characterize calcium handling in developing human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). To this end, real‐time polymerase chain reaction ...(PCR), immunocytochemistry, whole‐cell voltage‐clamp, and simultaneous patch‐clamp/laser scanning confocal calcium imaging and surface membrane labeling with di‐8‐aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC‐CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor‐2, and inositol‐1,4,5‐trisphosphate (IP3) receptors. Store calcium function was manifested as action‐potential‐induced calcium transients. Time‐to‐target plots showed that these action‐potential‐initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC‐CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine‐sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early‐stage (with the initiation of beating), intermediate‐stage (10 days post‐beating dpb), and late‐stage (30–40 dpb) hESC‐CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3‐releasable calcium pool was also demonstrated in the hESC‐CMs in experiments using caged‐IP3 photolysis and antagonist application (2 μM 2‐Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early‐stage hESC‐CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC‐CMs both for developmental studies and for the development of future myocardial cell replacement strategies.
Disclosure of potential conflicts of interest is found at the end of this article.
Human embryonic stem cell-derived cardiomyocytes (hES-CMs) are thought to recapitulate the embryonic development of heart
cells. Given the exciting potential of hES-CMs as replacement tissue in ...diseased hearts, we investigated the pharmacological
sensitivity and ionic current of mid-stage hES-CMs (20â35 days post plating). A high-resolution microelectrode array was used
to assess conduction in multicellular preparations of hES-CMs in spontaneously contracting embryoid bodies (EBs). TTX (10
μ m ) dramatically slowed conduction velocity from 5.1 to 3.2 cm s â1 while 100 μ m TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca 2+ channel blockers nifedipine or diltiazem (1 μ m ) had a negligible effect on conduction. These results suggested a prominent Na + channel current, and therefore we patch-clamped isolated cells to record Na + current and action potentials (APs). We found for isolated hES-CMs a prominent Na + current (244 ± 42 pA pF â1 at 0 mV; n = 19), and a hyperpolarization-activated current (HCN), but no inward rectifier K + current. In cell clusters, 3 μ m TTX induced longer AP interpulse intervals and 10 μ m TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca 2+ channel block) and 2 m m Cs + (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum
upstroke velocity (d V /d t max ) of 118 ± 14 V s â1 in control conditions; in contrast, partial block of Na + current significantly reduced stimulated d V /d t max (38 ± 15 V s â1 ). RT-PCR revealed Na V 1.5, Ca V 1.2, and HCN-2 expression but we could not detect Kir2.1. We conclude that hES-CMs at mid-range development express prominent
Na + current. The absence of background K + current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of Na V 1.5; thus, the Na V 1.5 Na + channel is important for initiating spontaneous excitability in hES-derived heart cells.
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