Christiaan C. Veerman

Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem?

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 cardiac sodium channel gene SCN5A and its gene product NaV1.5: Role in physiology and pathophysiology

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.

Slow Delayed Rectifier Potassium Current Blockade Contributes Importantly to Drug-Induced Long QT Syndrome

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 &agr;-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.Background —Drug-induced long QT syndrome (LQTS) is generally ascribed to inhibition of the cardiac rapid delayed rectifier potassium current, I Kr . Effects on the slow delayed rectifier potassium current I Ks are less recognized. Triggered by a patient who carried the K422T mutation in KCNQ1 (encoding the α-subunit of the I Ks channel), who presented with excessive QT prolongation and high serum levels of norfluoxetine, we investigated the effects of fluoxetine and its metabolite norfluoxetine on I Ks . Methods and Results —ECG data from mutation carriers and non-carriers 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 I Ks and I Kr demonstrated significant action potential prolongation, to which I Ks block contributed importantly. While the effects of the mutation per se were small, additional I Ks blockade by norfluoxetine resulted in more prominent QTc prolongation in mutation carriers than in non-carriers, demonstrating synergistic effects of innate and drug-induced I Ks blockade on QTc prolongation. Conclusions — I Ks blockade contributes importantly to drug-induced LQTS, especially when repolarization reserve is reduced. Drug safety tests might have to include screening for I Ks blockade.

IKs Blockade Contributes Importantly to Drug-Induced Long QT Syndrome

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 &agr;-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.Background —Drug-induced long QT syndrome (LQTS) is generally ascribed to inhibition of the cardiac rapid delayed rectifier potassium current, I Kr . Effects on the slow delayed rectifier potassium current I Ks are less recognized. Triggered by a patient who carried the K422T mutation in KCNQ1 (encoding the α-subunit of the I Ks channel), who presented with excessive QT prolongation and high serum levels of norfluoxetine, we investigated the effects of fluoxetine and its metabolite norfluoxetine on I Ks . Methods and Results —ECG data from mutation carriers and non-carriers 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 I Ks and I Kr demonstrated significant action potential prolongation, to which I Ks block contributed importantly. While the effects of the mutation per se were small, additional I Ks blockade by norfluoxetine resulted in more prominent QTc prolongation in mutation carriers than in non-carriers, demonstrating synergistic effects of innate and drug-induced I Ks blockade on QTc prolongation. Conclusions — I Ks blockade contributes importantly to drug-induced LQTS, especially when repolarization reserve is reduced. Drug safety tests might have to include screening for I Ks blockade.

The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical HeterogeneityNovelty and Significance

Rationale: Genome-wide association studies previously identified an association of rs9388451 at chromosome 6q22.3 (near HEY2) with Brugada syndrome. 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+/− (Hey2 heterozygous knockout) mice to dissect the underpinnings of the 6q22.31 association with Brugada syndrome. Methods and Results: We queried expression quantitative trait locus data acquired in 190 human left ventricular samples from the genotype-tissue expression consortium for cis-expression quantitative trait locus effects of rs9388451, which revealed an association between Brugada syndrome risk allele dosage and HEY2 expression (&bgr;=+0.159; P=0.0036). In the same transcriptomic data, we conducted genome-wide coexpression analysis for HEY2, which uncovered KCNIP2, encoding the &bgr;-subunit of the channel underlying the transient outward current (Ito), as the transcript most robustly correlating with HEY2 expression (&bgr;=+1.47; P=2×10−34). Transcript abundance of Hey2 and the Ito subunits Kcnip2 and Kcnd2, assessed by quantitative reverse transcription–polymerase chain reaction, was higher in subepicardium versus subendocardium in both left and right ventricles, with lower levels in Hey2+/− mice compared with wild type. Surface ECG measurements showed less prominent J waves in Hey2+/− mice compared with wild-type. In wild-type mice, patch-clamp electrophysiological studies on cardiomyocytes from right ventricle demonstrated a shorter action potential duration and a lower Vmax in subepicardium compared with subendocardium cardiomyocytes, which was paralleled by a higher Ito and a lower sodium current (INa) density in subepicardium versus subendocardium. These transmural differences were diminished in Hey2+/− mice because of changes in subepicardial cardiomyocytes. 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 Brugada syndrome through a HEY2-dependent alteration of ion channel expression across the cardiac ventricular wall.

hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities.

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.

Readthrough-Promoting Drugs Gentamicin and PTC124 Fail to Rescue Nav1.5 Function of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes Carrying Nonsense Mutations in the Sodium Channel Gene SCN5A

Background—Several compounds have been reported to induce translational readthrough of premature stop codons resulting in the production of full-length protein by interfering with ribosomal proofreading. Here we examined the effect of 2 of these compounds, gentamicin and PTC124, in human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes bearing nonsense mutations in the sodium channel gene SCN5A, which are associated with conduction disease and potential lethal arrhythmias. Methods and Results—We generated hiPSC from 2 patients carrying the mutations R1638X and W156X. hiPSC-derived cardiomyocytes from both patients recapitulated the expected electrophysiological phenotype, as evidenced by reduced Na+ currents and action potential upstroke velocities compared with hiPSC-derived cardiomyocytes from 2 unrelated control individuals. While we were able to confirm the readthrough efficacy of the 2 drugs in Human Embryonic Kidney 293 cells, we did not observe rescue of the electrophysiological phenotype in hiPSC-derived cardiomyocytes from the patients. Conclusions—We conclude that these drugs are unlikely to present an effective treatment for patients carrying the loss-of-function SCN5A gene mutations examined in this study.

Switch From Fetal to Adult SCN5A Isoform in Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes Unmasks the Cellular Phenotype of a Conduction Disease–Causing Mutation

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 (IN a) was mildly reduced in I230Thomo hiPSC‐CMs compared with control hiPSC‐CMs, while I230Thet hiPSC‐CMs displayed no reduction in IN a. 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 IN a 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 IN a, 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.

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

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 (IK1) 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 IK1 (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 IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

Response by Veerman et al to Letter Regarding Article, “The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity”

We welcome the opportunity to respond to the question raised by El-Battrawy et al in their letter concerning our study entitled “The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity.”1 In their letter, El-Battrawy et al state that the transmural differences observed in Hey2 +/− mice as compared with wild-type could be explained by the fact that the sodium current ( I Na) was measured at room temperature, whereas the transient outward potassium current ( I to) and action potential were evaluated at 36°C. The authors comment that sodium channel expression and sodium current density are modulated by temperature in Brugada Syndrome …