Effects of Acute Hypernatremia on the Electrophysiology of Single Human Ventricular Cardiomyocytes: An In Silico Study.

Background: Clinical and experimental data on the cardiac effects of acute hypernatremia are scarce and inconsistent. We aimed to determine and understand the effects of different levels of acute hypernatremia on the human ventricular action potential. Methods: We performed computer simulations using two different, very comprehensive models of the electrical activity of a single human ventricular cardiomyocyte, i.e., the Tomek-Rodriguez model following the O'Hara-Rudy dynamic (ORd) model and the Bartolucci-Passini-Severi model as published in 2020 (known as the ToR-ORd and BPS2020 models, respectively). Mild to extreme levels of hypernatremia were introduced into each model based on experimental data on the effects of hypernatremia on cell volume and individual ion currents. Results: In both models, we observed an increase in the intracellular sodium and potassium concentrations, an increase in the peak amplitude of the intracellular calcium concentration, a hyperpolarization of the resting membrane potential, a prolongation of the action potential, an increase in the maximum upstroke velocity, and an increase in the threshold stimulus current at all levels of hypernatremia and all stimulus rates tested. The magnitude of all of these effects was relatively small in the case of mild to severe hypernatremia but substantial in the case of extreme hypernatremia. The effects on the action potential were related to an increase in the sodium-potassium pump current, an increase in the sodium-calcium exchange current, a decrease in the rapid and slow delayed rectifier potassium currents, and an increase in the fast and late sodium currents. Conclusions: The effects of mild to severe hypernatremia on the electrical activity of human ventricular cardiomyocytes are relatively small. In the case of extreme hypernatremia, the effects are more pronounced, especially regarding the increase in threshold stimulus current.

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.

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.

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.

The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity.

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 (ß=+0.159; P=0.0036). In the same transcriptomic data, we conducted genome-wide coexpression analysis for HEY2, which uncovered KCNIP2, encoding the ß-subunit of the channel underlying the transient outward current (Ito), as the transcript most robustly correlating with HEY2 expression (ß=+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.

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.

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.

Computational analysis of the human sinus node action potential: model development and effects of mutations.

KEY POINTS: We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi-DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the 'funny current' (If ) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart-rate modulating drugs. ABSTRACT: The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi-DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller 'funny current' (If ) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss-of-function and gain-of-function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If , fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

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.