Action Potential Shape Research Articles

Using a Failing Human Ventricular Cardiomyocyte Model to Re-Evaluate Ca2+ Cycling, Voltage Dependence, and Spark Characteristics.

Previous studies have observed alterations in excitation-contraction (EC) coupling during end-stage heart failure that include action potential and calcium (Ca2+) transient prolongation and a reduction of the Ca2+ transient amplitude. Underlying these phenomena are the downregulation of potassium (K+) currents, downregulation of the sarcoplasmic reticulum Ca2+ ATPase (SERCA), increase Ca2+ sensitivity of the ryanodine receptor, and the upregulation of the sodium-calcium (Na=-Ca2+) exchanger. However, in human heart failure (HF), debate continues about the relative contributions of the changes in calcium handling vs. the changes in the membrane currents. To understand the consequences of the above changes, they are incorporated into a computational human ventricular myocyte HF model that can explore the contributions of the spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The reduction of transient outward K+ current (Ito) is the main membrane current contributor to the decrease in RyR2 open probability and L-type calcium channel (LCC) density which emphasizes its importance to phase 1 of the action potential (AP) shape and duration (APD). During current-clamp conditions, RyR2 hyperphosphorylation exhibits the least amount of Ca2+ release from the SR into the cytosol and SR Ca2+ fractional release during a dynamic slow-rapid-slow (0.5-2.5-0.5 Hz) pacing, but it displays the most abundant and more lasting Ca2+ sparks two-fold longer than a normal cell. On the other hand, under voltage-clamp conditions, HF by decreased SERCA and upregulated INCX show the least SR Ca2+ uptake and EC coupling gain, as compared to HF by hyperphosphorylated RyR2s. Overall, this study demonstrates that the (a) combined effect of SERCA and NCX, and the (b) RyR2 dysfunction, along with the downregulation of the cardiomyocyte's potassium currents, could substantially contribute to Ca2+ mishandling at the spark level that leads to heart failure.

Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1–S6). Silent Kv (KvS), includes Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1–9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4–S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.

Optimization of an anatomically and electrically detailed rodent subthalamic nucleus neuron model.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective treatment for Parkinson's disease, but its mechanisms of action remain unclear. Detailed multicompartment computational models of STN neurons are often used to study how DBS electric fields modulate the neurons. However, currently available STN neuron models have some limitations in their biophysical realism. In turn, the goal of this study was to update a detailed rodent STN neuron model originally developed by Gillies and Willshaw in 2006. Our design requirements consisted of explicitly representing an axon connected to the neuron and updating the ion channel distributions based on the experimental literature to match established electrophysiological features of rodent STN neurons. We found that adding an axon to the STN neuron model substantially altered its firing characteristics. We then used a genetic algorithm to optimize biophysical parameters of the model. The updated model exhibited spontaneous firing, action potential shape, hyperpolarization response, and frequency-current curve that aligned well with experimental recordings from STN neurons. Subsequently, we evaluated the general compatibility of the updated biophysics by applying them to 26 different STN neuron morphologies derived from three-dimensional anatomical reconstructions. The different morphologies affected the firing behavior of the model, but the updated biophysics were robustly capable of maintaining the desired electrophysiological features. The new STN neuron model developed in this work offers a valuable tool for studying STN neuron firing properties and may find application in simulating STN local field potentials and analyzing the effects of STN DBS.NEW & NOTEWORTHY This study presents an anatomically and biophysically realistic rodent STN neuron model. The work showcases the use of a genetic algorithm to optimize the model parameters. We noted a substantial influence of the axon on the electrophysiological characteristics of STN neurons. The updated model offers a valuable tool to investigate the firing of STN neurons and their modulation by intrinsic and/or extrinsic factors.

The interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers

Abstract Funding Acknowledgements None. Multi-electrode arrays (MEA) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using a MEA are like extracellular electrograms recorded from ventricular myocardium using conventional electrodes. Nevertheless, different criteria are often used to interpret field potential and extracellular electrogram, which hampers correct interpretation and translation to the patient. To validate criteria for extracellular electrograms analysis applicable to field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using a MEA and simultaneously recorded action potentials using sharp micro-electrodes. In parallel we recreated our experimental setting in silico and performed simulations. We demonstrate that the amplitude of the local RS-complex of a field potential correlated with conduction velocity, but only in the lower ranges of conduction velocities. The time of the peak of the T-wave of the field potentials correlated well with APD90 and the time of the steepest upslope with APD50. By injecting current into the monolayer, we could record a local extracellular action potential (LEAP). The LEAP was of low amplitude and highly depolarized, and the shape differed markedly from the shape of the local action potential. Criteria for interpreting extra cellular electrograms should also be applied to field potentials. This will provide a strong basis for analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes especially at low conduction velocities. A LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes.

Real-time (re)shaping of cardiac action potentials by an opto-electronic feedback control system in the multicellular setting

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council Starting Grant and Consolidator Grant to Daniël A Pijnappels Background The membrane potential (Vm) in cardiomyocytes fulfills essential regulatory roles in various biological functions, including not only action potential (AP) propagation and mechanical contraction, but also homeostatic regulation. In-depth studies into these roles have been severely hampered by the lack of research methods allowing full control over Vm, especially in multicellular cardiac preparations. Purpose We aimed to unlock new research possibilities by the development of an experimental system (APqr) capable of full Vm control, including instantaneous modulation of AP morphology on a multicellular levelin the multicellular setting. Methods Monolayers of immortalized human atrial myocytes (hiAMs, n=7) were genetically modified to express the blue light-activatable cation channel Cheriff and the red light-sensitive inward chloride pump Jaws for depolarizing and re- or hyperpolarizing effects, respectively. Real-time Vm readout was obtained by patch clamp electrophysiology. Deviations of Vm from reference values were calculated by a custom closed-coop controller. A 470-nm and a 617-nm LED were modulated by the controller in a Vm deviation-dependent manner, selectively activating Cheriff or Jaws. Electrical disturbances were introduced by application of 4-aminopyridien (4AP, 200 µM) or a preprogrammed blue-light pulse creating abnormal cation influx during the repolarization phase of the hiAM AP. Results Average AP durations at 90% repolarization (APD90) increased by 82.4 ms following 4AP application and by 362.5 ms in the presence of light-induced abnormal cation influx compared to control conditions. APqr reduced these APD90 differences to 1.9 ms and 4.6 ms on average, with Vm deviations less than 2.5 mV in 74.7% and 94.4% of the time, respectively. APqr could also be applied for the enforcement of arbitrary AP shapes. In these experiments, reference Vm values consisted of APs recorded from hiAM monolayers exposed to drugs with APD-prolonging (4AP) or shortening (carbachol) effects. APqr was able to enforce these reference APs on hiAM monolayers with high accuracy, with Vm deviations less than 2.5 mV in 95.8% and 94.4% of the time, respectively. Conclusions APqr preserves AP morphologies in the presence of electrical perturbations of different origin without any prior knowledge of the disturbance and enforces arbitrary AP morphologies with high accuracy in an immediate and self-regulatory manner. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory roles of the membrane potential in health and disease.

Human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulate contribution of the slowly activating delayed rectifier currents IKs to repolarization in the human atrium.

Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) could be a helpful tool to study the physiology and diseases of the human atrium. To fulfil this expectation, the electrophysiology of hiPSC-aCM should closely resemble the situation in the human atrium. Data on the contribution of the slowly activating delayed rectifier currents (IKs) to repolarization are lacking for both human atrium and hiPSC-aCM. Human atrial tissues were obtained from patients with sinus rhythm (SR) or atrial fibrillation (AF). Currents were measured in human atrial cardiomyocytes (aCM) and compared with hiPSC-aCM and used to model IKs contribution to action potential (AP) shape. Action potential was recorded by sharp microelectrodes. HMR-1556 (1 µM) was used to identify IKs and to estimate IKs contribution to repolarization. Less than 50% of hiPSC-aCM and aCM possessed IKs. Frequency of occurrence, current densities, activation/deactivation kinetics, and voltage dependency of IKs did not differ significantly between hiPSC-aCM and aCM, neither in SR nor AF. β-Adrenoceptor stimulation with isoprenaline did not increase IKs neither in aCM nor in hiPSC-aCM. In tissue from SR, block of IKs with HMR-1556 did not lengthen the action potential duration, even when repolarization reserve was reduced by block of the ultra-rapid repolarizing current with 4-aminopyridine or the rapidly activating delayed rectifier potassium outward current with E-4031. I Ks exists in hiPSC-aCM with biophysics not different from aCM. As in adult human atrium (SR and AF), IKs does not appear to relevantly contribute to repolarization in hiPSC-aCM.

Computational modeling to study the impact of changes in Nav1.8 sodium channel on neuropathic pain.

Nav1.8 expression is restricted to sensory neurons; it was hypothesized that aberrant expression and function of this channel at the site of injury contributed to pathological pain. However, the specific contributions of Nav1.8 to neuropathic pain are not as clear as its role in inflammatory pain. The aim of this study is to understand how Nav1.8 present in peripheral sensory neurons regulate neuronal excitability and induce various electrophysiological features on neuropathic pain. To study the effect of changes in sodium channel Nav1.8 kinetics, Hodgkin-Huxley type conductance-based models of spiking neurons were constructed using the NEURON v8.2 simulation software. We constructed a single-compartment model of neuronal soma that contained Nav1.8 channels with the ionic mechanisms adapted from some existing small DRG neuron models. We then validated and compared the model with our experimental data from in vivo recordings on soma of small dorsal root ganglion (DRG) sensory neurons in animal models of neuropathic pain (NEP). We show that Nav1.8 is an important parameter for the generation and maintenance of abnormal neuronal electrogenesis and hyperexcitability. The typical increased excitability seen is dominated by a left shift in the steady state of activation of this channel and is further modulated by this channel's maximum conductance and steady state of inactivation. Therefore, modified action potential shape, decreased threshold, and increased repetitive firing of sensory neurons in our neuropathic animal models may be orchestrated by these modulations on Nav1.8. Computational modeling is a novel strategy to understand the generation of chronic pain. In this study, we highlight that changes to the channel functions of Nav1.8 within the small DRG neuron may contribute to neuropathic pain.

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

Characteristics of A-type voltage-gated K+ currents expressed on sour-sensing type III taste receptor cells in mice

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K+ currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K+ currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K+ currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K+ currents which were completely inhibited by 10 mM TEA, whereas IP3R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K+ currents were 17.9 ± 4.5 (n = 17) and − 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K+ channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K+ channel phosphorylation likely affects the signal transduction of taste.

Oxytocin Modifies the Excitability and the Action Potential Shape of the Hippocampal CA1 GABAergic Interneurons.

Oxytocin (OT) is a neuropeptide that modulates social-related behavior and cognition in the central nervous system of mammals. In the CA1 area of the hippocampus, the indirect effects of the OT on the pyramidal neurons and their role in information processing have been elucidated. However, limited data are available concerning the direct modulation exerted by OT on the CA1 interneurons (INs) expressing the oxytocin receptor (OTR). Here, we demonstrated that TGOT (Thr4,Gly7-oxytocin), a selective OTR agonist, affects not only the membrane potential and the firing frequency but also the neuronal excitability and the shape of the action potentials (APs) of these INs in mice. Furthermore, we constructed linear mixed-effects models (LMMs) to unravel the dependencies between the AP parameters and the firing frequency, also considering how TGOT can interact with them to strengthen or weaken these influences. Our analyses indicate that OT regulates the functionality of the CA1 GABAergic INs through different and independent mechanisms. Specifically, the increase in neuronal firing rate can be attributed to the depolarizing effect on the membrane potential and the related enhancement in cellular excitability by the peptide. In contrast, the significant changes in the AP shape are directly linked to oxytocinergic modulation. Importantly, these alterations in AP shape are not associated with the TGOT-induced increase in neuronal firing rate, being themselves critical for signal processing.

Kinetics and functional consequences of BK channels activation by N-type Ca2+ channels in the dendrite of mouse neocortical layer-5 pyramidal neurons.

The back-propagation of an action potential (AP) from the axon/soma to the dendrites plays a central role in dendritic integration. This process involves an intricate orchestration of various ion channels, but a comprehensive understanding of the contribution of each channel type remains elusive. In this study, we leverage ultrafast membrane potential recordings (Vm) and Ca2+ imaging techniques to shed light on the involvement of N-type voltage-gated Ca2+ channels (VGCCs) in layer-5 neocortical pyramidal neurons' apical dendrites. We found a selective interaction between N-type VGCCs and large-conductance Ca2+-activated K+ channels (BK CAKCs). Remarkably, we observe that BK CAKCs are activated within a mere 500 μs after the AP peak, preceding the peak of the Ca2+ current triggered by the AP. Consequently, when N-type VGCCs are inhibited, the early broadening of the AP shape amplifies the activity of other VGCCs, leading to an augmented total Ca2+ influx. A NEURON model, constructed to replicate and support these experimental results, reveals the critical coupling between N-type and BK channels. This study not only redefines the conventional role of N-type VGCCs as primarily involved in presynaptic neurotransmitter release but also establishes their distinct and essential function as activators of BK CAKCs in neuronal dendrites. Furthermore, our results provide original functional validation of a physical interaction between Ca2+ and K+ channels, elucidated through ultrafast kinetic reconstruction. This insight enhances our understanding of the intricate mechanisms governing neuronal signaling and may have far-reaching implications in the field.

Interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers.

Multielectrode arrays (MEAs) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using an MEA are like extracellular electrograms recorded from the myocardium using conventional electrodes. Nevertheless, different criteria are used to interpret field potentials and extracellular electrograms, which hamper correct interpretation and translation to the patient. To validate the criteria for interpretation of field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using an MEA and simultaneously recorded action potentials using sharp microelectrodes. In parallel, we recreated our experimental setting in silico and performed simulations. We show that the amplitude of the local RS complex of a field potential correlated with conduction velocity in silico but not in vitro. The peak time of the T wave in field potentials exhibited a strong correlation with APD90 while the steepest upslope correlated well with APD50. However, this relationship only holds when the T wave displayed a biphasic pattern. Next, we simulated local extracellular action potentials (LEAPs). The shape of the LEAP differed markedly from the shape of the local action potential, but the final duration of the LEAP coincided with APD90. Criteria for interpretation of extracellular electrograms should be applied to field potentials. This will provide a strong basis for the analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes. Finally, a LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes and is superior to field potential duration in estimating APD90.NEW & NOTEWORTHY We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

Rapid neurostimulation at the micron scale with an optically controlled thermal-capture technique.

Precise control of cellular temperature at the microscale is crucial for developing novel neurostimulation techniques. Here, the effect of local heat on the electrophysiological properties of primary neuronal cultures and HEK293 cells at the subcellular level using a cutting-edge micrometer-scale thermal probe, the diamond heater-thermometer (DHT), is studied. A new mode of local heat action on a living cell, thermal-capture mode (TCM), is discovered using the DHT probe. In TCM, the application of a 50 °C temperature step induces a great increase in cellular response, allowing the cell to be thermally captured and depolarized by up to 20 mV. This thermal effect is attributed to local phase changes in the phospholipid membrane, enabling precise and reproducible modulation of cell activity. The TCM is shown to open up new opportunities for thermal cell stimulation. DHT reliably triggers action potentials (APs) in neurons at rates up to 30 Hz, demonstrating the ability to control cell excitability with millisecond and sub-millisecond resolution. AP shape is modulated by local heat as well. The ability to precisely control the AP shape and rate via thermal-capture mode opens new avenues for non-invasive, localized neurostimulation techniques, particularly in controlling neuron excitability.

Prediction of Dexterous Finger Forces with Forearm Rotation using Motoneuron Discharges.

Motor unit (MU) discharge information obtained via electromyogram (EMG) decomposition can be used to decode dexterous multi-finger movement intention for neural-machine interfaces (NMI). However, the variation of the motor unit action potential (MUAP) shape resulted from forearm rotation leads to the decreased performance of EMG decomposition, especially under the real-time condition and then the degradation of motion decoding accuracy. The object of this study was to develop a method to realize the accurate extraction of MU discharge information across forearm pronated/supinated positions in the real-time condition for dexterous multi-finger force prediction. The FastICA-based EMG decomposition technique was used and the proposed method obtained multiple separation vectors for each MU at different forearm positions in the initialization phase. Under the real-time condition, the MU discharge information was extracted adaptively using the separation vector extracted at the nearest forearm position. As comparison, the previous method that utilized a single constant separation vector to extract MU discharges across forearm positions and the conventional method that utilized the EMG amplitude information were also performed. The results showed that the proposed method obtained a significantly better performance compared with the other two methods, manifested in a larger coefficient of determination (R2) and a smaller root mean squared error (RMSE) between the predicted and recorded force. Our results demonstrated the feasibility and the effectiveness of the proposed method to extract MU discharge information during forearm rotation for dexterous force prediction under the real-time conditions. Further development of the proposed method could potentially promote the application of the EMG decomposition technique for continuous dexterous motion decoding in a realistic NMI application scenario.

Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

An opto-electronic control system for real-time complete (re)shaping of cardiac action potentials on a multicellular scale

Abstract Background Cardiomyocytes maintain a membrane potential (Vm), which fulfills essential regulatory roles in various biological functions, including not only action potential (AP) propagation and mechanical contraction, but also homeostatic regulation. In-depth studies into these roles have been severely hampered by the lack of research methods allowing full control over Vm, especially in multicellular cardiac preparations. Purpose We aimed to unlock new research possibilities by the development of an experimental system (APqr) capable of full Vm control, including instantaneous modulation of AP morphology on a multicellular level. Methods Immortalized human atrial myocytes (hiAMs) grown in monolayer format (n=7) were genetically modified to express the blue light-activatable cation channel Cheriff and the red light-sensitive inward chloride pump Jaws for depolarizing and hyperpolarizing effects, respectively. Real-time Vm readout was obtained by patch clamp electrophysiology. Dynamic Vm control was achieved by a custom closed-coop controller calculating Vm deviations from reference values. A 470-nm and a 617-nm LED were modulated by the controller in a Vm deviation-dependent manner, selectively activating Cheriff and Jaws. To explore the feasibility of AP restoration, electrical disturbances were introduced by non-selective potassium channel blockade (4-aminopyridine, 4AP, 200 µM) or by a preprogrammed blue-light pulse creating abnormal cation influx during the repolarization phase of the hiAM AP. Results 4AP induced a 82.4 ± 27.8 ms increase of AP duration at 90% repolarization (APD90) values in hiAM monolayers. Using APs recorded under control conditions (CTL) as reference, the APqr maneuver reduced this APD90 difference to 1.9 ± 3.1 ms. Similarly, APqr suppressed the effects of light-induced abnormal cation influx on APD90 (CTL: 342.9 ± 51.7 ms, blue-light pulse: 700.8 ± 64.5 ms, APqr: 338.3 ± 68 ms) effectively, with Vm deviation less than 2.5 mV in 96.7% of the time. APqr could also be applied for the enforcement of arbitrary AP shapes. In these experiments, the Vm references consisted of AP shapes of hiAM monolayers exposed to drugs with APD-prolonging (4AP) or shortening (carbachol) effects. APqr was able to enforce these reference APs, that were distinctly different from the endogenous AP shapes, on hiAM monolayers with great accuracy, with Vm deviations less than 2.5 mV in 95.8% and 94.4% of the time, respectively. Conclusions APqr restores AP morphologies in the presence of electrical perturbations of different origin without any prior knowledge about the disturbance and enforces arbitrary AP morphologies with high accuracy in an immediate and self-regulatory manner. Collectively, these results set the stage for in-depth investigations into the regulatory roles of Vm in healthy and diseased cardiomyocytes via the application of such real-time opto-electronic control systems.Real-time (re)shaping of cardiac APs

A critical role of retinoic acid concentration for the induction of a fully human-like atrial action potential phenotype in hiPSC-CM

Retinoic acid (RA) induces an atrial phenotype in human induced pluripotent stem cells (hiPSCs), but expression of atrium-selective currents such as the ultrarapid (IKur) and acetylcholine-stimulated K+ current is variable and less than in the adult human atrium. We suspected methodological issues and systematically investigated the concentration dependency of RA. RA treatment increased IKur concentration dependently from 1.1± 0.54pA/pF (0 RA) to 3.8± 1.1, 5.8± 2.5, and 12.2± 4.3 at 0.01, 0.1, and 1μM, respectively. Only 1μM RA induced enough IKur to fully reproduce human atrial action potential (AP) shape and a robust shortening of APs upon carbachol. We found that sterile filtration caused substantial loss of RA. We conclude that 1μM RA seems to be necessary and sufficient to induce a full atrial AP shape in hiPSC-CM in EHT format. RA concentrations are prone to methodological issues and may profoundly impact the success of atrial differentiation.

Action potential morphology affects T-wave symmetry (simulation study)

BackgroundAssessing T-wave symmetry in addition to QT subintervals measurements can provide novel independent data about ventricular repolarization abnormalities linked with arrhythmogenesis. However, the causes of the changes of T-wave symmetry are not completely understood. In silico studies showed that the more symmetrical T-waves were associated with shorter action potential duration (APD) and larger dispersion of ventricular repolarization (DOR). The aim of present simulation was to study the association between T-wave symmetry and action potential (AP) shape. MethodsECGs were simulated using a cellular automata model shaped as a ventricular wall segment, and two biophysically-detailed models of ventricular AP – the rabbit and the human. The symmetry ratio (SR) was calculated as a T-wave onset-peak to peak-end area ratio. The individual and combined effects of APD, DOR and AP shape on SR were simulated. To study the effect of AP shape, different APs from triangulated to rectangular were simulated. ResultsThe simulations showed that AP shape along with APD and DOR contributes much to T-wave symmetry. APs with a flat phase 3 (triangulated) produced asymmetrical T-waves (SR ≥ 1.5) in all simulations, except the shortest APD range. APs with a rapid phase 3 (rectangular) were associated with more symmetrical T-waves (SR ≤ =1) both at the short and the long APDs. ConclusionSR marker in combination with the standard ECG parameters (QT interval, Tpeak-Tend interval) may be useful to identify the proarrhythmic triangulated AP shape.

Disease severity, arrhythmogenesis, and fibrosis are related to longer action potentials in tetralogy of Fallot.

Arrhythmias may originate from surgically unaffected right ventricular (RV) regions in patients with tetralogy of Fallot (TOF). We aimed to investigate action potential (AP) remodelling and arrhythmia susceptibility in RV myocardium of patientswith repaired and with unrepaired TOF, identify possible correlations with clinical phenotype and myocardial fibrosis, and compare findings with data from patients with atrial septal defect (ASD), a less severe congenital heart disease. Intracellular AP were recorded ex vivo in RV outflow tract samples from 22 TOF and three ASD patients. Arrhythmias were provoked by superfusion with solutions containing reduced potassium and barium chloride, or isoprenaline. Myocardial fibrosis was quantified histologically and associations between clinical phenotype, AP shape, tissue arrhythmia propensity, and fibrosis were examined. Electrophysiological abnormalities (arrhythmias, AP duration [APD] alternans, impaired APD shortening at increased stimulation frequencies) were generally present in TOF tissue, even from infants, but rare or absent in ASD samples. More severely diseased and acyanotic patients, pronounced tissue susceptibility to arrhythmogenesis, and greater fibrosis extent were associated with longer APD. In contrast, APD was shorter in tissue from patients with pre-operative cyanosis. Increased fibrosis and repaired-TOF status were linked to tissue arrhythmia inducibility. Functional and structural tissue remodelling may explain arrhythmic activity in TOF patients, even at a very young age. Surprisingly, clinical acyanosis appears to be associated with more severe arrhythmogenic remodelling. Further research into the clinical drivers of structural and electrical myocardial alterations, and the relation between them, is needed to identify predictive factors for patients at risk.

Whole-cell patch-clamp recording and parameters.

The patch-clamp technique represents an electrophysiology type of method. This is one of several insightful approaches with five major configurations, namely a loose patch, cell-attached (also known as on-cell), whole-cell, inside-out, and outside-out modes. The patch-clamp method is more advanced compared to classical electrophysiology since it elucidates single-channel activation in a tiny portion of the membrane in addition to action potential (AP), junction potential (JP), endplate potential (EP), electrical coupling between two adjacent cells via Gap junction hemi-channels, excitatory/inhibitory postsynaptic potentials, and resting membrane potential (RMP). In fact, a malfunction of only one channel or even one component will alter AP amplitude or duration in vitro. If parameters are inferred appropriately and recordings are performed properly, the patch-clamp trace readouts and results are robust. The main hallmarks of currents via voltage-dependent calcium (Cav), hyperpolarization-activated cyclic nucleotide gated non-selective cation (HCN), inwardly rectifying potassium (Kir), voltage-dependent potassium (Kv), and voltage-dependent sodium (Nav) channels are similar and tractable among cells even when they are derived from evolutionary distinct organs and species. However, the size of the membrane area, where the functional subunits reside, and current magnitudes vary among cells of the same type. Therefore, dividing current magnitudes by cell capacitance- current density enables the estimate of functional and active channels relative to recorded cytoplasmic membrane area. Since the patch-clamp recordings can be performed in both current- and voltage-clamp modes, the action potential or spike durations can be adequately elucidated. Sometimes, optical methods are preferred to patch-clamp electrophysiology, but the obtained signals and traces are not robust. Finally, not only an alternans of AP durations, but also that of 'action potential shape' is observed with electrophysiology.