Injection of IK1 through dynamic clamp can make all the difference in patch-clamp studies on hiPSC-derived cardiomyocytes.

Human-induced stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool for studying development, pharmacology, and (inherited) arrhythmias. Unfortunately, hiPSC-CMs are depolarized and spontaneously active, even the working cardiomyocyte subtypes such as atrial- and ventricular-like hiPSC-CMs, in contrast to the situation in the atria and ventricles of adult human hearts. Great efforts have been made, using many different strategies, to generate more mature, quiescent hiPSC-CMs with more close-to-physiological resting membrane potentials, but despite promising results, it is still difficult to obtain hiPSC-CMs with such properties. The dynamic clamp technique allows to inject a current with characteristics of the inward rectifier potassium current (IK1), computed in real time according to the actual membrane potential, into patch-clamped hiPSC-CMs during action potential measurements. This results in quiescent hiPSC-CMs with a close-to-physiological resting membrane potential. As a result, action potential measurements can be performed with normal ion channel availability, which is particularly important for the physiological functioning of the cardiac SCN5A-encoded fast sodium current (INa). We performed in vitro and in silico experiments to assess the beneficial effects of the dynamic clamp technique in dissecting the functional consequences of the SCN5A-1795insD+/- mutation. In two separate sets of patch-clamp experiments on control hiPSC-CMs and on hiPSC-CMs with mutations in ACADVL and GNB5, we assessed the value of dynamic clamp in detecting delayed afterdepolarizations and in investigating factors that modulate the resting membrane potential. We conclude that the dynamic clamp technique has highly beneficial effects in all of the aforementioned settings and should be widely used in patch-clamp studies on hiPSC-CMs while waiting for the ultimate fully mature hiPSC-CMs.

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data.

These days, in vitro functional analysis of gene variants is becoming increasingly important for risk stratification of cardiac ion channelopathies. So far, such risk stratification has been applied to SCN5A, KCNQ1, and KCNH2 gene variants associated with Brugada syndrome and long QT syndrome types 1 and 2, respectively, but risk stratification of HCN4 gene variants related to sick sinus syndrome has not yet been performed. HCN4 is the gene responsible for the hyperpolarization-activated 'funny' current If, which is an important modulator of the spontaneous diastolic depolarization underlying the sinus node pacemaker activity. In the present study, we carried out a risk classification assay on those loss-of-function mutations in HCN4 for which in vivo as well as in vitro data have been published. We used the in vitro data to compute the charge carried by If (Qf) during the diastolic depolarization phase of a prerecorded human sinus node action potential waveform and assessed the extent to which this Qf predicts (1) the beating rate of the comprehensive Fabbri-Severi model of a human sinus node cell with mutation-induced changes in If and (2) the heart rate observed in patients carrying the associated mutation in HCN4. The beating rate of the model cell showed a very strong correlation with Qf from the simulated action potential clamp experiments (R2 = 0.95 under vagal tone). The clinically observed minimum or resting heart rates showed a strong correlation with Qf (R2 = 0.73 and R2 = 0.71, respectively). While a translational perspective remains to be seen, we conclude that action potential clamp on transfected cells, without the need for further voltage clamp experiments and data analysis to determine individual biophysical parameters of If, is a promising tool for risk stratification of sinus bradycardia due to loss-of-function mutations in HCN4. In combination with an If blocker, this tool may also prove useful when applied to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from mutation carriers and non-carriers.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and ß-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and ß-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking ß-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

The Antidepressant Paroxetine Reduces the Cardiac Sodium Current.

A considerable amount of literature has been published on antidepressants and cardiac ion channel dysfunction. The antidepressant paroxetine has been associated with Brugada syndrome and long QT syndrome, albeit on the basis of conflicting findings. The cardiac voltage-gated sodium channel (NaV1.5) is related to both of these syndromes, suggesting that paroxetine may have an effect on this channel. In the present study, we therefore carried out patch clamp experiments to examine the effect of paroxetine on human NaV1.5 channels stably expressed in human embryonic kidney 293 (HEK-293) cells as well as on action potentials of isolated rabbit left ventricular cardiomyocytes. Additionally, computer simulations were conducted to test the functional effects of the experimentally observed paroxetine-induced changes in the NaV1.5 current. We found that paroxetine led to a decrease in peak NaV1.5 current in a concentration-dependent manner with an IC50 of 6.8 ± 1.1 µM. In addition, paroxetine caused a significant hyperpolarizing shift in the steady-state inactivation of the NaV1.5 current as well as a significant increase in its rate of inactivation. Paroxetine (3 µM) affected the action potential of the left ventricular cardiomyocytes, significantly decreasing its maximum upstroke velocity and amplitude, both of which are mainly regulated by the NaV1.5 current. Our computer simulations demonstrated that paroxetine substantially reduces the fast sodium current of human left ventricular cardiomyocytes, thereby slowing conduction and reducing excitability in strands of cells, in particular if conduction and excitability are already inhibited by a loss-of-function mutation in the NaV1.5 encoding SCN5A gene. In conclusion, paroxetine acts as an inhibitor of NaV1.5 channels, which may enhance the effects of loss-of-function mutations in SCN5A.

Acetylcholine Reduces L-Type Calcium Current without Major Changes in Repolarization of Canine and Human Purkinje and Ventricular Tissue.

Vagal nerve stimulation (VNS) holds a strong basis as a potentially effective treatment modality for chronic heart failure, which explains why a multicenter VNS study in heart failure with reduced ejection fraction is ongoing. However, more detailed information is required on the effect of acetylcholine (ACh) on repolarization in Purkinje and ventricular cardiac preparations to identify the advantages, risks, and underlying cellular mechanisms of VNS. Here, we studied the effect of ACh on the action potential (AP) of canine Purkinje fibers (PFs) and several human ventricular preparations. In addition, we characterized the effects of ACh on the L-type Ca2+ current (ICaL) and AP of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and performed computer simulations to explain the observed effects. Using microelectrode recordings, we found a small but significant AP prolongation in canine PFs. In the human myocardium, ACh slightly prolonged the AP in the midmyocardium but resulted in minor AP shortening in subepicardial tissue. Perforated patch-clamp experiments on hiPSC-CMs demonstrated that 5 µM ACh caused an ≈15% decrease in ICaL density without changes in gating properties. Using dynamic clamp, we found that under blocked K+ currents, 5 µM ACh resulted in an ≈23% decrease in AP duration at 90% of repolarization in hiPSC-CMs. Computer simulations using the O'Hara-Rudy human ventricular cell model revealed that the overall effect of ACh on AP duration is a tight interplay between the ACh-induced reduction in ICaL and ACh-induced changes in K+ currents. In conclusion, ACh results in minor changes in AP repolarization and duration of canine PFs and human ventricular myocardium due to the concomitant inhibition of inward ICaL and outward K+ currents, which limits changes in net repolarizing current and thus prevents major changes in AP repolarization.

Carbamazepine Increases the Risk of Sudden Cardiac Arrest by a Reduction of the Cardiac Sodium Current.

Aim: To assess the risk of sudden cardiac arrest (SCA) associated with the use of carbamazepine (CBZ) and establish the possible underlying cellular electrophysiological mechanisms. Methods: The SCA risk association with CBZ was studied in general population cohorts using a case-control design (n = 5,473 SCA cases, 21,866 non-SCA controls). Effects of 1-100 µM CBZ on action potentials (APs) and individual membrane currents were determined in isolated rabbit and human cardiomyocytes using the patch clamp technique. Results: CBZ use was associated with increased risk of SCA compared with no use (adjusted odds ratio 1.90 [95% confidence interval: 1.12-3.24]). CBZ reduced the AP upstroke velocity of rabbit and human cardiomyocytes, without prominent changes in other AP parameters. The reduction occurred at ≥30 µM and was frequency-dependent with a more pronounced reduction at high stimulus frequencies. The cardiac sodium current (INa) was reduced at ≥30 µM; this was accompanied by a hyperpolarizing shift in the voltage-dependency of inactivation. The recovery from inactivation was slower, which is consistent with the more pronounced AP upstroke velocity reduction at high stimulus frequencies. The main cardiac K+ and Ca2+ currents were unaffected, except reduction of L-type Ca2+ current by 100 µM CBZ. Conclusion: CBZ use is associated with an increased risk of SCA in the general population. At concentrations of 30 µM and above, CBZ reduces AP upstroke velocity and INa in cardiomyocytes. Since the concentration of 30 µM is well within the therapeutic range (20-40 µM), we conclude that CBZ increases the risk of SCA by a reduction of the cardiac INa.

shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele.

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs.

Acetylcholine Reduces IKr and Prolongs Action Potentials in Human Ventricular Cardiomyocytes.

Vagal nerve stimulation (VNS) has a meaningful basis as a potentially effective treatment for heart failure with reduced ejection fraction. There is an ongoing VNS randomized study, and four studies are completed. However, relatively little is known about the effect of acetylcholine (ACh) on repolarization in human ventricular cardiomyocytes, as well as the effect of ACh on the rapid component of the delayed rectifier K+ current (IKr). Here, we investigated the effect of ACh on the action potential parameters in human ventricular preparations and on IKr in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Using standard microelectrode technique, we demonstrated that ACh (5 µM) significantly increased the action potential duration in human left ventricular myocardial slices. ACh (5 µM) also prolonged repolarization in a human Purkinje fiber and a papillary muscle. Optical mapping revealed that ACh increased the action potential duration in human left ventricular myocardial slices and that the effect was dose-dependent. Perforated patch clamp experiments demonstrated action potential prolongation and a significant decrease in IKr by ACh (5 µM) in hiPSC-CMs. Computer simulations of the electrical activity of a human ventricular cardiomyocyte showed an increase in action potential duration upon implementation of the experimentally observed ACh-induced changes in the fully activated conductance and steady-state activation of IKr. Our findings support the hypothesis that ACh can influence the repolarization in human ventricular cardiomyocytes by at least changes in IKr.

HCN4 current during human sinoatrial node-like action potentials.

BACKGROUND: Despite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated 'funny' current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. OBJECTIVE: To study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. METHODS: HCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500-1500 ms cycle length, in absence or presence of forskolin to mimic ß-adrenergic stimulation and a -15 mV command potential offset to mimic atrial load. RESULTS: Forskolin significantly increased the fully-activated HCN4 current density at -140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. CONCLUSIONS: If is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, ß-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Patch-Clamp Recordings of Action Potentials From Human Atrial Myocytes: Optimization Through Dynamic Clamp.

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings. Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K+ current (IK1), and consequently with a regular, non-depolarized resting membrane potential (RMP), through "dynamic clamp". Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic IK1 to generate a regular RMP. The injected IK1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics. Results: Without any current injection, RMPs ranged from -9.6 to -86.2 mV in 58 cells. In depolarized cells (RMP positive to -60 mV), RMP could be set at -80 mV using IK1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods (p < 0.05) and was shortest with constant current injection and longest with injection of IK1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD90) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic IK1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used. Conclusion: Injection of a synthetic IK1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.

Dynamic Clamp in Electrophysiological Studies on Stem Cell-Derived Cardiomyocytes-Why and How?

ABSTRACT: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are supposed to be a good human-based model, with virtually unlimited cell source, for studies on mechanisms underlying cardiac development and cardiac diseases, and for identification of drug targets. However, a major drawback of hPSC-CMs as a model system, especially for electrophysiological studies, is their depolarized state and associated spontaneous electrical activity. Various approaches are used to overcome this drawback, including the injection of "synthetic" inward rectifier potassium current (IK1), which is computed in real time, based on the recorded membrane potential ("dynamic clamp"). Such injection of an IK1-like current results in quiescent hPSC-CMs with a nondepolarized resting potential that show "adult-like" action potentials on stimulation, with functional availability of the most important ion channels involved in cardiac electrophysiology. These days, dynamic clamp has become a widely appreciated electrophysiological tool. However, setting up a dynamic clamp system can still be laborious and difficult, both because of the required hardware and the implementation of the dedicated software. In the present review, we first summarize the potential mechanisms underlying the depolarized state of hPSC-CMs and the functional consequences of this depolarized state. Next, we explain how an existing manual patch clamp setup can be extended with dynamic clamp. Finally, we shortly validate the extended setup with atrial-like and ventricular-like hPSC-CMs. We feel that dynamic clamp is a highly valuable tool in the field of cellular electrophysiological studies on hPSC-CMs and hope that our directions for setting up such dynamic clamp system may prove helpful.

Ultrarapid Delayed Rectifier K+ Channelopathies in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5-15% of AF patients have a mutation in a cardiac gene, including mutations in KCNA5, encoding the Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant IKur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native IKur, measured as current sensitive to 50 µM 4-aminopyridine, was 1.88 ± 0.49 (mean ± SEM, n = 17) and 0.26 ± 0.26 pA/pF (n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.

Self-restoration of cardiac excitation rhythm by anti-arrhythmic ion channel gating.

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a "funny current" and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Genetic variation in GNB5 causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current (IK,ACh).

Mutations in GNB5, encoding the G-protein ß5 subunit (Gß5), 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 Gß5 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 GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as IK,ACh]. 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 (IK,ACh) 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 GNB5 mutations.This article has an associated First Person interview with the first author of the paper.

Aquaporin Channels in the Heart-Physiology and Pathophysiology.

Mammalian aquaporins (AQPs) are transmembrane channels expressed in a large variety of cells and tissues throughout the body. They are known as water channels, but they also facilitate the transport of small solutes, gasses, and monovalent cations. To date, 13 different AQPs, encoded by the genes AQP0-AQP12, have been identified in mammals, which regulate various important biological functions in kidney, brain, lung, digestive system, eye, and skin. Consequently, dysfunction of AQPs is involved in a wide variety of disorders. AQPs are also present in the heart, even with a specific distribution pattern in cardiomyocytes, but whether their presence is essential for proper (electro)physiological cardiac function has not intensively been studied. This review summarizes recent findings and highlights the involvement of AQPs in normal and pathological cardiac function. We conclude that AQPs are at least implicated in proper cardiac water homeostasis and energy balance as well as heart failure and arsenic cardiotoxicity. However, this review also demonstrates that many effects of cardiac AQPs, especially on excitation-contraction coupling processes, are virtually unexplored.

Long QT Syndrome and Sinus Bradycardia-A Mini Review.

Congenital long-QT syndrome (LQTS) is an inherited cardiac disorder characterized by the prolongation of ventricular repolarization, susceptibility to Torsades de Pointes (TdP), and a risk for sudden death. Various types of congenital LQTS exist, all due to specific defects in ion channel-related genes. Interestingly, almost all of the ion channels affected by the various types of LQTS gene mutations are also expressed in the human sinoatrial node (SAN). It is therefore not surprising that LQTS is frequently associated with a change in basal heart rate (HR). However, current data on how the LQTS-associated ion channel defects result in impaired human SAN pacemaker activity are limited. In this mini-review, we provide an overview of known LQTS mutations with effects on HR and the underlying changes in expression and kinetics of ion channels. Sinus bradycardia has been reported in relation to a large number of LQTS mutations. However, the occurrence of both QT prolongation and sinus bradycardia on a family basis is almost completely limited to LQTS types 3 and 4 (LQT3 and Ankyrin-B syndrome, respectively). Furthermore, a clear causative role of this sinus bradycardia in cardiac events seems reserved to mutations underlying LQT3.

KV4.3 Expression Modulates NaV1.5 Sodium Current.

In cardiomyocytes, the voltage-gated transient outward potassium current (Ito) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the Ito carrying KV4.3 channel, have been associated with Brugada syndrome (BrS). While the role of Ito in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased Ito may directly affect cardiac conduction. However, the effects of an increased Ito on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of KV4.3 overexpression on NaV1.5 current and consequent sodium channel availability. We found that overexpression of KV4.3 protein in HEK293 cells stably expressing NaV1.5 (HEK293-NaV1.5 cells) significantly reduced NaV1.5 current density without affecting its kinetic properties. In addition, KV4.3 overexpression decreased AP upstroke velocity in HEK293-NaV1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of KV4.3 could not be explained by alterations in total NaV1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in KV4.3 current and concurrent decrease in NaV1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that KV4.3 directly impacts on NaV1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.

Sinus Bradycardia in Carriers of the SCN5A-1795insD Mutation: Unraveling the Mechanism through Computer Simulations.

The SCN5A gene encodes the pore-forming α-subunit of the ion channel that carries the cardiac fast sodium current (INa). The 1795insD mutation in SCN5A causes sinus bradycardia, with a mean heart rate of 70 beats/min in mutation carriers vs. 77 beats/min in non-carriers from the same family (lowest heart rate 41 vs. 47 beats/min). To unravel the underlying mechanism, we incorporated the mutation-induced changes in INa into a recently developed comprehensive computational model of a single human sinoatrial node cell (Fabbri-Severi model). The 1795insD mutation reduced the beating rate of the model cell from 74 to 69 beats/min (from 49 to 43 beats/min in the simulated presence of 20 nmol/L acetylcholine). The mutation-induced persistent INa per se resulted in a substantial increase in beating rate. This gain-of-function effect was almost completely counteracted by the loss-of-function effect of the reduction in INa conductance. The further loss-of-function effect of the shifts in steady-state activation and inactivation resulted in an overall loss-of-function effect of the 1795insD mutation. We conclude that the experimentally identified mutation-induced changes in INa can explain the clinically observed sinus bradycardia. Furthermore, we conclude that the Fabbri-Severi model may prove a useful tool in understanding cardiac pacemaker activity in humans.

Cellular Mechanisms of Sinus Node Dysfunction in Carriers of the SCN5A-E161K Mutation and Role of the H558R Polymorphism.

Background: Carriers of the E161K mutation in the SCN5A gene, encoding the NaV1.5 pore-forming α-subunit of the ion channel carrying the fast sodium current (INa), show sinus bradycardia and occasional exit block. Voltage clamp experiments in mammalian expression systems revealed a mutation-induced 2.5- to 4-fold reduction in INa peak current density as well as a +19 mV shift and reduced steepness of the steady-state activation curve. The highly common H558R polymorphism in NaV1.5 limits this shift to +13 mV, but also introduces a -10 mV shift in steady-state inactivation. Aim: We assessed the cellular mechanism by which the E161K mutation causes sinus node dysfunction in heterozygous mutation carriers as well as the potential role of the H558R polymorphism. Methods: We incorporated the mutation-induced changes in INa into the Fabbri-Severi model of a single human sinoatrial node cell and the Maleckar et al. human atrial cell model, and carried out simulations under control conditions and over a wide range of acetylcholine levels. Results: In absence of the H558R polymorphism, the E161K mutation increased the basic cycle length of the sinoatrial node cell from 813 to 866 ms. In the simulated presence of 10 and 25 nM acetylcholine, basic cycle length increased from 1027 to 1131 and from 1448 to 1795 ms, respectively. The increase in cycle length was the result of a significant slowing of diastolic depolarization. The mutation-induced reduction in INa window current had reduced the contribution of the mutant component of INa to the net membrane current during diastolic depolarization to effectively zero. Highly similar results were obtained in presence of the H558R polymorphism. Atrial excitability was reduced, both in absence and presence of the H558R polymorphism, as reflected by an increase in threshold stimulus current and a concomitant decrease in capacitive current of the atrial cell. Conclusion: We conclude that the experimentally identified mutation-induced changes in INa can explain the clinically observed sinus bradycardia and potentially the occasional exit block. Furthermore, we conclude that the common H558R polymorphism does not significantly alter the effects of the E161K mutation and can thus not explain the reduced penetrance of the E161K mutation.