Cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are increasingly used to model cardiac disease, test drug efficacy and for safety pharmacology. Nevertheless, a major hurdle to more ...extensive use is their immaturity and similarity to fetal rather than adult cardiomyocytes. Here, we provide an overview of the strategies currently being used to increase maturation in culture, which include prolongation of time in culture, exposure to electrical stimulation, application of mechanical strain, growth in three-dimensional tissue configuration, addition of non-cardiomyocytes, use of hormones and small molecules, and alteration of the extracellular environment. By comparing the outcomes of these studies, we identify the approaches most likely to improve functional maturation of hPSC-CMs in terms of their electrophysiology and excitation-contraction coupling.
The gene SCN5A encodes the main cardiac sodium channel NaV1.5. This channel predominates the cardiac sodium current, INa, which underlies the fast upstroke of the cardiac action potential. As such, ...it plays a crucial role in cardiac electrophysiology. Over the last 60years a tremendous amount of knowledge regarding its function at the electrophysiological and molecular level has been acquired. Furthermore, genetic studies have shown that mutations in SCN5A are associated with multiple cardiac diseases (e.g. Brugada syndrome, Long QT syndrome, conduction disease and cardiomyopathy), while genetic variation in the general population has been associated with differences in cardiac conduction and risk of arrhythmia through genome wide association studies. In this review we aim to give an overview of the current knowledge (and the gaps therein) on SCN5A and NaV1.5.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. ...Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (
) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial
(computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic
through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon
injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic
. In conclusion, the dynamic clamp-based approach of
injection has broad applicability for detailed AP measurements in hiPSC-CMs.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Brugada syndrome (BrS) is a rare cardiac rhythm disorder associated with sudden cardiac death. Mutations in the sodium channel gene SCN5A are found in ~20% of cases while mutations in other genes ...collectively account for <5%. In the remaining patients the genetic defect and the underlying pathogenic mechanism remain obscure. To provide insight into the mechanism of BrS in individuals without identified mutations, we here studied electrophysiological properties of cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) from 3 BrS patients who tested negative for mutations in the known BrS-associated genes. Patch clamp studies revealed no differences in sodium current (INa) in hiPSC-CMs from the 3 BrS patients compared to 2 unrelated controls. Moreover, action potential upstroke velocity (Vmax), reflecting INa, was not different between hiPSC-CMs from the BrS patients and the controls. hiPSC-CMs harboring the BrS-associated SCN5A-1795insD mutation exhibited a reduction in both INa and Vmax, demonstrating our ability to detect reduced sodium channel function. hiPSC-CMs from one of the BrS lines demonstrated a mildly reduced action potential duration, however, the transient outward potassium current (Ito) and the L-type calcium current (ICa,L), both implicated in BrS, were not different compared to the controls. Our findings indicate that ion channel dysfunction, in particular in the cardiac sodium channel, may not be a prerequisite for BrS.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
RATIONALE:Genome-wide association studies previously identified an association of rs9388451 at chromosome 6q22.3 (near HEY2) with Brugada syndrome (BrS). The causal gene and underlying mechanism ...remain unresolved.
OBJECTIVE:We used an integrative approach entailing transcriptomic studies in human hearts and electrophysiological studies in Hey2 heterozygous knockout mice (Hey2) to dissect the underpinnings of the 6q22.31 association with BrS.
METHODS AND RESULTS:We queried expression quantitative trait locus (eQTL) data acquired in 190 human left ventricular (LV) samples from the Genotype-Tissue Expression (GTEx) consortium for cis-eQTL effects of rs9388451 which revealed an association between BrS risk allele dosage and HEY2 expression (β=+0.159; P=0.0036). In the same transcriptomic data, we conducted genome-wide co-expression analysis for HEY2 which uncovered KCNIP2, encoding the β-subunit of the channel underlying the transient outward current (Ito), as the transcript most robustly correlating with HEY2 expression (β=+1.47; P=2X10-). Transcript abundance of HEY2 and the Ito subunits Kcnip2 and Kcnd2, assessed by qRT-PCR, was higher in subepicardium (epi) vs. subendocardium (endo) in both left (LV) and right (RV) ventricles, with lower levels in HEY2 heterozygous knockout (Hey2) mice compared to wildtype (WT). Surface ECG measurements showed less prominent J-waves in Hey2 mice compared to WT. In WT mice, patch-clamp electrophysiological studies on cardiac myocytes from RV demonstrated a shorter AP duration and a lower Vmax in epi compared to endo cardiac myocytes, which was paralleled by a higher Ito and a lower INa density in epi vs. endo. These transmural differences were diminished in Hey2 mice due to changes in subepicardial cardiac myocytes.
CONCLUSIONS:This study uncovers a role of HEY2 in the normal transmural electrophysiological gradient in the ventricle and provides compelling evidence that genetic variation at 6q22.31 (rs9388451) is associated with BrS through a HEY2-dependent alteration of ion channel expression across the cardiac ventricular wall.
Background
Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) can recapitulate features of ion channel mutations causing inherited rhythm disease. However, the lack of maturity of ...these cells is considered a significant limitation of the model. Prolonged culture of hiPSC‐CMs promotes maturation of these cells. We studied the electrophysiological effects of the I230T mutation in the sodium channel gene SCN5A in hiPSC‐CMs generated from a homozygous (I230Thomo) and a heterozygous (I230Thet) individual from a family with recessive cardiac conduction disease. Since the I230T mutation occurs in the developmentally regulated “adult” isoform of SCN5A, we investigated the relationship between the expression fraction of the adult SCN5A isoform and the electrophysiological phenotype at different time points in culture.
Methods and Results
After a culture period of 20 days, sodium current (INa) was mildly reduced in I230Thomo hiPSC‐CMs compared with control hiPSC‐CMs, while I230Thet hiPSC‐CMs displayed no reduction in INa. This coincided with a relatively high expression fraction of the “fetal” SCN5A isoform compared with the adult isoform as measured by quantitative polymerase chain reaction. Following prolonged culture to 66 days, the fraction of adult SCN5A isoform increased; this was paralleled by a marked decrease in INa in I230Thomo hiPSC‐CMs, in line with the severe clinical phenotype in homozygous patients. At this time in culture, I230Thet hiPSC‐CMs displayed an intermediate loss of INa, compatible with a gene dosage effect.
Conclusions
Prolonged culture of hiPSC‐CMs leads to an increased expression fraction of the adult sodium channel isoform. This new aspect of electrophysiological immaturity should be taken into account in studies that focus on the effects of SCN5A mutations in hiPSC‐CMs.
Mutations in
, encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying ...bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the
p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel I(KACh); also known as I
. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current (
) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying
mutations.This article has an associated First Person interview with the first author of the paper.
BACKGROUND—Drug-induced long QT syndrome is generally ascribed to inhibition of the cardiac rapid delayed rectifier potassium current (IKr). Effects on the slow delayed rectifier potassium current ...(IKs) are less recognized. Triggered by a patient who carried the K422T mutation in KCNQ1 (encoding the α-subunit of the IKs channel), who presented with excessive QT prolongation and high serum levels of norfluoxetine, we investigated the effects of fluoxetine and its metabolite norfluoxetine on IKs.
METHODS AND RESULTS—ECG data from mutation carriers and noncarriers revealed that the K422T mutation per se had mild clinical effects. Patch clamp studies, performed on HEK293 cells, showed that heterozygously expressed K422T KCNQ1/KCNE1 channels had a positive shift in voltage dependence of activation and an increase in deactivation rate. Fluoxetine and its metabolite norfluoxetine both inhibited KCNQ1/KCNE1 current, with norfluoxetine being the most potent. Moreover, norfluoxetine increased activation and deactivation rates. Computer simulations of the effects of norfluoxetine on IKs and IKr demonstrated significant action potential prolongation, to which IKs block contributed importantly. Although the effects of the mutation per se were small, additional IKs blockade by norfluoxetine resulted in more prominent QTc prolongation in mutation carriers than in noncarriers, demonstrating synergistic effects of innate and drug-induced IKs blockade on QTc prolongation.
CONCLUSIONS—IKs blockade contributes importantly to drug-induced long QT syndrome, especially when repolarization reserve is reduced. Drug safety tests might have to include screening for IKs blockade.
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