Phase Of Action Potential Research Articles

In silico analysis of ventricular action potential with a current-voltage-time representation: Thresholds, membrane resistance, repolarization reserve.

The waveform of ventricular action potential (AP) is a key determinant of the cardiac cycle, a marker of beating pathophysiology, and a target for anti-arrhythmic drug design. The information contained in the waveform, though, is limited to the actual dynamics of the AP under consideration. By measuring quasi-instantaneous current-voltage relationships during repolarization, I propose a three-dimensional representation of the ventricular AP which includes potential dynamic responses that the beat can show when electrically perturbed. This representation is described in the case of a numerically reconstructed ventricular AP, but it can be, at least partially, derived in real cardiomyocytes. Simulation allows to disclose the potentialities and the limitations of the approach, that can be extended to any non-cardiac AP. By reporting, at any AP time, the ion current available within the physiological membrane potential range at that time, the representation makes all together available: (1) refractory period, (2) thresholds for eliciting full or calcium-driven APs, (3) threshold for all-or-none repolarization, (4) membrane resistance during repolarization, (5) the safety of membrane repolarization. It provides further evidence of a negative membrane resistance during the late phase of ventricular AP and a quantitative estimate of repolarization reserve (RR), key determinants of repolarization dynamics.

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.

Biophysical properties of NaV1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes expressed higher atrial- and sinoatrial-specific markers (MYL7, CACNA1D) and lower ventricular-specific markers (MYL2, CACNA1C, GJA1) than ventricular cardiomyocytes. The amplitude, duration, and steady-state phase of APs in atrial cardiomyocytes decreased, and had a shape similar to that of mature atrial cardiomyocytes. Interestingly, NaV1.5 channels in atrial cardiomyocytes exhibited lower mRNA transcripts and protein expression, which could explain the lower current densities recorded by patch-clamp. Moreover, Na+ currents exhibited differences in activation and inactivation parameters. These differences could be explained by an increase in SCN2B regulatory subunit expression and a decrease in SCN1B and SCN4B regulatory subunit expressions. Our results show that a RA treatment made it possible to obtain atrial cardiomyocytes and investigate differences in NaV1.5 channel properties between ventricular- and atrial-like cells.

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

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 β-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and β-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 β-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.

Suppression of transmembrane sodium currents on the freshly isolated hippocampal neuron cell with continuous infrared light

Physiotherapeutic effects of infrared lasers have been proved in clinic. These infrared-based regulations of the bioelectrical activities can roughly be classified into enhancement and suppression of action potential (AP), which are described by sodium (Na) and potassium (K) transmembrane current equations, named as Hodgkin and Huxley (HH)-model. The enhancement effect is able to evoke or strengthen the AP when infrared light is applied. Its corresponding mechanism is commonly ascribed to the changes of the cell membrane capacitance, which is transiently increased in response to the infrared radiation. The distinctive feature of the suppression effect is to inhibit or reduce the AP by the designed protocols of infrared radiation. However, its mechanism presents more complexity than that in enhancement cases. HH-model describes how the Na current determines the initial phase of AP. So, the enhancement and suppression of AP can be also ascribed to the regulations of the corresponding Na currents. Here, a continuous infrared light at the wavelength of 980[Formula: see text]nm (CIS-980) was employed to stimulate a freshly isolated hippocampal neuron in vitro and a suppression effect on the Na currents of the neuron cell was observed. Both Na and K currents, which are named as whole cell currents, were simultaneously recorded with the cell membrane capacitance current by using a patch clamp combined with infrared irradiation. The results demonstrated that the CIS-980 was able to reversibly increase the capacitance currents, completely suppressed Na currents, but little changed K currents, which forms the steady outward whole cell currents and plays a major role on the AP repolarization. A confirmation experiment was designed and carried out by synchronizing tens of milliseconds of infrared stimulation on the same kinds of hippocampal neuron cells. After the blocked K channel, a reduction of Na current amplitude was still recorded. This proved that infrared suppression of Na current was irrelevant to K channel. A membrane capacitance mediation process was preliminarily proposed to explain the Na channel suppression process.

Improved Ca2+ release synchrony following selective modification of Itof and phase 1 repolarization in normal and failing ventricular myocytes

Loss of ventricular action potential (AP) early phase 1 repolarization may contribute to the impaired Ca2+ release and increased risk of sudden cardiac death in heart failure. Therefore, restoring AP phase 1 by augmenting the fast transient outward K+ current (Itof) might be beneficial, but direct experimental evidence to support this proposition in failing cardiomyocytes is limited. Dynamic clamp was used to selectively modulate the contribution of Itof to the AP and Ca2+ transient in both normal (guinea pig and rabbit) and in failing rabbit cardiac myocytes. Opposing native Itof in non-failing rabbit myocytes increased Ca2+ release heterogeneity, late Ca2+ sparks (LCS) frequency and AP duration. (APD). In contrast, increasing Itof in failing myocytes and guinea pig myocytes (the latter normally lacking Itof) increased Ca2+ transient amplitude, Ca2+ release synchrony, and shortened APD. Computer simulations also showed faster Ca2+ transient decay (mainly due to fewer LCS), decreased inward Na+/Ca2+ exchange current and APD. When the Itof conductance was increased to ~0.2 nS/pF in failing cells (a value slightly greater than seen in typical human epicardial myocytes), Ca2+ release synchrony improved and AP duration decreased slightly. Further increases in Itof can cause Ca2+ release to decrease as the peak of the bell-shaped ICa-voltage relationship is passed and premature AP repolarization develops. These results suggest that there is an optimal range for Itof enhancement that may support Ca2+ release synchrony and improve electrical stability in heart failure with the caveat that uncontrolled Itof enhancement should be avoided.

Abstract 10814: Prolongation of the Refractory Period is a Key Mechanism of Optogenetic Termination of Atrial Fibrillation in Mouse Hearts

Backgrounds: Optogenetics is a new technique that employs light-sensitive channels and manipulates the cell membrane potential. Although painless defibrillation of reentry arrhythmias such as atrial fibrillation (AF) using optogenetics has been reported, the mechanism has not been fully elucidated because of the difficulty to evaluate the action potential (AP) modification by in/ex-vivo light illumination. Purpose: Our aim is to clarify the mechanism of optogenetic defibrillation of AF in a mouse heart by voltage-sensitive optical mapping. For this purpose, we employed a novel approach using brief light illumination combined with a systematic electrical stimulation protocol. Methods and Results: Hearts from transgenic mice expressing channelrhodopsin-2 in atrial cardiomyocytes were explanted and perfused with tyrode’s solution containing low K + concentration (2.7mM) and K ACh -channel activator carbachol (2μM). Atrial tachyarrhythmia induced by burst pacing was terminated by blue light (470nm) illumination with a pulse duration of 10 ms and a light intensity of 16 mW/mm 2 with a probability of 64.7 ± 8.1% (n=9). The termination rate depended on the pulse duration and light intensity. Then, we examined whether brief light illumination (10 ms, 6mW/mm 2 ) at different time phases from the rise of the AP (phase 0) to the repolarization (phase 4) modify AP shape and prolong the effective refractory period (ERP) using voltage-sensitive optical mapping. Brief light illumination prolonged AP duration and ERP in all time phases. The degree of ERP prolongation was dependent on the pulse duration, but not on the light intensity above 1 mW/mm 2 . Finally, we measured the attenuation of blue light intensity through the atrial tissue and observed that the intensity decreased by 38.1 ± 3.5% at 10mW/mm 2 on 3 mm 2 circular area(n=6), suggesting that high-intensity light also modulates the APs of atrial cardiomyocytes in deeper layers. Conclusion: Our results directly demonstrated that brief light illumination prolonged the atrial refractory period depending on pulse duration in a perfused heart and suggests the prolongation of the refractory period is a key mechanism of optogenetic termination of AF.

Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation-contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.

Insulin acts as an atypical KCNQ1/KCNE1‐current activator and reverses long QT in insulin‐resistant aged rats by accelerating the ventricular action potential repolarization through affecting the β3‐adrenergic receptor signaling pathway

Insufficient-heart function is associated with myocardial insulin resistance in the elderly, particularlyassociated with long-QT, in a dependency on dysfunctional KCNQ1/KCNE1-channels. So, we aimed to examine the contribution of alterations in KCNQ1/KCNE1-current (IKs ) to the aging-related remodeling of the heart as well as the role of insulin treatment on IKs in the aged rats. Prolonged late-phase action potential (AP) repolarization of ventricular cardiomyocytes from insulin-resistant 24-month-old rats was significantly reversed by in vitro treatment of insulin or PKG inhibitor (in vivo, as well) via recovery in depressed IKs . Although the protein level of either KCNQ1 or KCNE1 in cardiomyocytes was not affected with aging, PKG level was significantly increased in those cells. The inhibited IKs in β3 -ARs-stimulated cells could be reversed with a PKG inhibitor, indicating the correlation between PKG-activation and β3 -ARs activation. Furthermore, in vivo treatment of aged rats, characterized by β3 -ARs activation, with either insulin or a PKG inhibitor for 2 weeks provided significant recoveries in IKs , prolonged late phases of APs, prolonged QT-intervals, and low heart rates without no effect on insulin resistance. In vivo insulin treatment provided also significant recovery in increased PKG and decreased PIP2 level, without the insulin effect on the KCNQ1 level in β3 -ARs overexpressed cells. The inhibition of IKs in aged-rat cardiomyocytes seems to be associated with activated β3 -ARs dependent remodeling in the interaction between KCNQ1 and KCNE1. Significant recoveries in ventricular-repolarization of insulin-treated aged cardiomyocytes via recovery in IKs strongly emphasize two important issues: (1) IKs can be a novel target in aging-associated remodeling in the heart and insulin may be a cardioprotective agent in the maintenance of normal heart function during the aging process. (2) This study is one of the first to demonstrate insulin'sbenefits on long-QT in insulin-resistant aged rats by accelerating the ventricular AP repolarization through reversing the depressed IKs via affecting the β3 -ARs signaling pathway and particularly affecting activated PKG.

Spinal Cord Injury Significantly Alters the Properties of Reticulospinal Neurons: I. Biophysical Properties, Firing Patterns, Excitability, and Synaptic Inputs.

Following spinal cord injury (SCI) for larval lampreys, descending axons of reticulospinal (RS) neurons regenerate, and locomotor function gradually recovers. In the present study, the electrophysiological properties of uninjured (left)-injured (right) pairs of large, identified RS neurons were compared following rostral, right spinal cord hemi-transections (HTs). First, changes in firing patterns of injured RS neurons began in as little as 2–3 days following injury, these changes were maximal at ~2–3 weeks (wks), and by 12–16 wks normal firing patterns were restored for the majority of neurons. Second, at ~2–3 wks following spinal cord HTs, injured RS neurons displayed several significant changes in properties compared to uninjured neurons: (a) more hyperpolarized VREST; (b) longer membrane time constant and larger membrane capacitance; (c) increased voltage and current thresholds for action potentials (APs); (d) larger amplitudes and durations for APs; (e) higher slope for the repolarizing phase of APs; (f) virtual absence of some afterpotential components, including the slow afterhyperpolarization (sAHP); (g) altered, injury-type firing patterns; and (h) reduced average and peak firing (spiking) frequencies during applied depolarizing currents. These altered properties, referred to as the “injury phenotype”, reduced excitability and spiking frequencies of injured RS neurons compared to uninjured neurons. Third, artificially injecting a current to add a sAHP waveform following APs for injured neurons or removing the sAHP following APs for uninjured neurons did not convert these neurons to normal firing patterns or injury-type firing patterns, respectively. Fourth, trigeminal sensory-evoked synaptic responses recorded from uninjured and injured pairs of RS neurons were not significantly different. Following SCI, injured lamprey RS neurons displayed several dramatic changes in their biophysical properties that are expected to reduce calcium influx and provide supportive intracellular conditions for axonal regeneration.

Hyperexcitability of Nucleus Tractus Solitarii Neurons in the Streptozotocin‐Induced Model of Alzheimer's Disease

Besides the stereotypical problems of memory and cognition in patients with Alzheimer's Disease (AD), another highly prevalent symptom is of respiratory origin. We have repeatedly shown blunted respiratory responses to hypoxia in the streptozotocin (STZ) -induced AD model, which is similar to sleep disordered breathing found in nearly 80% of AD patients. Our current study focuses on the electrophysiological changes within the nucleus tractus solitarii (nTS), a brainstem area known to control respiratory function. Using male Sprague-Dawley rats (n = 15 rats, 240±8 g), we induced AD symptoms with microinjections of 2 mg/kg Streptozotocin (STZ) into the lateral cerebral ventricles. Control animals received 0.9 mM citrate buffer as vehicle control (CTL). The disease model was verified using the Morris water maze to test for memory dysfunction. We recorded nTS neuronal responses via whole cell patch clamp. Action potential (AP) discharge upon current-stimulation was higher in STZ-AD when compared to control. Also, spontaneous spike activity (no stimulation) was elevated. These data corroborate earlier results using a higher (2x1.5 mg) STZ dosage. Spontaneous post-synaptic currents in the absence of stimulation resemble nTS network activity. The number of spontaneous events in nTS neurons of the STZ-AD group was surprisingly similar to CTL in the current study. On the other hand, network activity mirrors the hyperexcitability seen from our current injection when using the higher STZ dosage. While there was no appreciable change in resting membrane potential, AP threshold of STZ-AD nTS cells shifted towards a more negative membrane potential, overall contributing to increased excitability in these cells. AP phase plane plots in STZ-AD had a significantly smaller overshoot with a reduced velocity of up and down stroke, possibly indicating altered function of voltage-gated sodium channels. Analysis of transient K+ currents (A-type) revealed a slight shift to higher activation voltages (from -20 mV [CTL] to 0 mV [STZ-AD]) in STZ-AD. This shift favors AP generation and may contribute to a lower spike threshold in STZ-AD. There were no changes in the steady state K+ currents between groups. In summary, nTS cells of the STZ-AD group showed increased excitability that may involve gating properties of voltage-gated sodium channels and A-type potassium channels. The observed neurophysiological changes in the nTS may be part of the neuronal correlate for increased breathing frequency and blunted (ceiling effect) respiratory responses to hypoxia in the STZ-AD model.

Interrelated In Vitro Mechanisms of Sibutramine-Induced Cardiotoxicity.

Consumption of illicit pharmaceutical products containing sibutramine has been reported to cause cardiovascular toxicity problems. This study aimed to demonstrate the toxicity profile of sibutramine, and thereby provide important implications for the development of more effective strategies in both clinical approaches and drug design studies. Action potentials (APs) were determined from freshly isolated ventricular cardiomyocytes with whole-cell configuration of current clamp as online. The maximum amplitude of APs (MAPs), the resting membrane potential (RMP), and AP duration from the repolarization phases were calculated from original records. The voltage-dependent K+-channel currents (IK) were recorded in the presence of external Cd2+ and both inward and outward parts of the current were calculated, while their expression levels were determined with qPCR. The levels of intracellular free Ca2+ and H+ (pHi) as well as reactive oxygen species (ROS) were measured using either a ratiometric micro-spectrofluorometer or confocal microscope. The mechanical activity of isolated hearts was observed with Langendorff-perfusion system. Acute sibutramine applications (10-8-10-5M) induced significant alterations in both MAPs and RMP as well as the repolarization phases of APs and IK in a concentration-dependent manner. Sibutramine (10μM) induced Ca2+-release from the sarcoplasmic reticulum under either electrical or caffeine stimulation, whereas it depressed left ventricular developed pressure with a marked decrease in the end-diastolic pressure. pHi inhibition by sibutramine supports the observed negative alterations in contractility. Changes in mRNA levels of different IK subunits are consistent with the acute inhibition of the repolarizing IK, affecting AP parameters, and provoke the cardiotoxicity.

Impact of ISK Voltage and Ca2+/Mg2+-Dependent Rectification on Cardiac Repolarization

Cardiac small conductance Ca2+-activated K+ (SK) channels are activated solely by Ca2+, but the SK current (ISK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca2+/Mg2+ block. Thus, Ca2+ has a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the effect of ISK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of ISK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca2+] reached ∼10 μM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] decreased further. We used a six-state gating model combined with intrinsic and Ca2+/Mg2+-dependent rectification to simulate ISK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of ISK recording during AP clamp showing that intrinsic rectification limits ISK at high Vm during the early and plateau phase of APs. Furthermore, the initial rise of Ca2+ transients activates, but higher [Ca2+] blocks SK channels, yielding a transient outward-like ISK trajectory. During the decay phase of Ca2+, the Ca2+-dependent block is released, causing ISK to rise again and contribute to repolarization. Therefore, ISK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.

BK channel openers NS1619 and NS11021 reverse hydrogen peroxide-induced membrane potential changes in skeletal muscle

Large conductance calcium-activated potassium (BK) channels play a crucial role in the repolarization and after-hyperpolarization phases of the cell membrane. The channel openers are also used in treatment of some diseases, including hypo/hyperkalemic periodic paralysis. However, little is known about the effects of BK channels and the channel activators on membrane potentials in skeletal muscle. In addition, the effects of reactive oxygen species (ROS) on BK channels in skeletal muscle are also unknown. Therefore, the aim of this study was to determine the effects of BK channel openers and ROS on membrane potentials in skeletal muscle fibers. For this purpose, resting membrane potentials and action potentials (AP) of frog gastrocnemius muscles were recorded in the presence of commonly used BK channel openers NS1619 and NS11021, H2O2 (a type of ROS), and both using intracellular microelectrode technique. The channel activators significantly and dose-dependently decreased amplitude and increased rise time of AP but did not impact repolarization. The presence of H2O2 plus NS1619 or NS11021 resulted in significant change because the channel openers completely reversed the deleterious effects of hydrogen peroxide on the repolarization phase of AP in skeletal muscle fibers. In the present study, the contributions of BK channel activation and the modulatory role of H2O2 on membrane potentials was demonstrated in skeletal muscle fibers, for the first time. Moreover, it should be noted that BK channel openers should be used in the treatment of reactive oxygen species-induced skeletal muscle diseases.

Role of SK Current Rectification in Shaping Action Potential of Ventricular Cardiomyocytes

The role of cardiac small conductance Ca2+-activated K+ (SK) channels in shaping the action potential (AP) of ventricular cardiomyocytes remains undefined. Two processes underlie inward rectification of SK current (ISK), an intrinsic voltage-dependent rectification caused by positively charged amino acids at the inner pore, and a divalent ion block. The latter process gives Ca2+ a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the role of ISK rectification on AP in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform, and developed a computer model of SK channel with rectification features. The typical profile of ISK during AP-clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point where submembrane [Ca2+] reached ∼10 μM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] continued to decrease. We used a six state gating model combined with intrinsic rectification and divalent block to simulate ISK and investigated the relative contributions of the two types of rectification to the AP shape. This SK channel model replicates key features of ISK recording during AP-clamp. The simulation shows that the rise of Ca2+ during Ca2+ transients activates SK channel opening, but higher [Ca2+] blocks channels, resulting in transient outward-like trajectory of ISK. During the decay phase of Ca2+, Ca2+-dependent block is released causing ISK to contribute to repolarization. The intrinsic rectification characteristics limit ISK during the early and plateau phase of APs to prevent excessive APD shortening. In conclusion, ISK is an important repolarizing current and the rectification characteristics of SK channel determines its impact on early, plateau and repolarization phases of APs.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Characterization of changes in the configuration of action potentials in the mouse, guinea pig, and pig sinoauricular node after application of channel blockers of the rapid and slow delayed rectifier potassium currents.

We hypothesized that the repolarization phase of action potentials (APs) in mammals with large body mass and high cardiac output could not be reliably controlled by only one of the delayed rectifier potassium IK current components. To test this hypothesis experimentally, we performed a comparative study of the response of AP phases to the rapid IKr channels blocker E-4031 and slow IKs blocker chromanol 293B in APs spontaneously generated in strips of sinoauricular (SA) tissue from mouse, guinea pig, and pig hearts. Application of a slow channels blocker chromanol 293B caused a decrease of Aps generation frequency in SA area strips from mouse, guinea-pig and pig by 5.3, 16, and 18% compared to the control. Treatment with the IKr blocker E-4031 caused a significant reduction of APs generation frequency in the mouse, guinea pig, and pig SA strips by 24, 26, and 36%, respectively, compared to the control values. These results suggest that the rapid IKr current is the key component responsible for AP generation in sinoauricular node cells of the pig heart.

Transmural Autonomic Regulation of Cardiac Contractility at the Intact Heart Level.

The relationship between cardiac excitability and contractility depends on when Ca2+ influx occurs during the ventricular action potential (AP). In mammals, it is accepted that Ca2+ influx through the L-type Ca2+ channels occurs during AP phase 2. However, in murine models, experimental evidence shows Ca2+ influx takes place during phase 1. Interestingly, Ca2+ influx that activates contraction is highly regulated by the autonomic nervous system. Indeed, autonomic regulation exerts multiple effects on Ca2+ handling and cardiac electrophysiology. In this paper, we explore autonomic regulation in endocardial and epicardial layers of intact beating mice hearts to evaluate their role on cardiac excitability and contractility. We hypothesize that in mouse cardiac ventricles the influx of Ca2+ that triggers excitation–contraction coupling (ECC) does not occur during phase 2. Using pulsed local field fluorescence microscopy and loose patch photolysis, we show sympathetic stimulation by isoproterenol increased the amplitude of Ca2+ transients in both layers. This increase in contractility was driven by an increase in amplitude and duration of the L-type Ca2+ current during phase 1. Interestingly, the β-adrenergic increase of Ca2+ influx slowed the repolarization of phase 1, suggesting a competition between Ca2+ and K+ currents during this phase. In addition, cAMP activated L-type Ca2+ currents before SR Ca2+ release activated the Na+-Ca2+ exchanger currents, indicating Cav1.2 channels are the initial target of PKA phosphorylation. In contrast, parasympathetic stimulation by carbachol did not have a substantial effect on amplitude and kinetics of endocardial and epicardial Ca2+ transients. However, carbachol transiently decreased the duration of the AP late phase 2 repolarization. The carbachol-induced shortening of phase 2 did not have a considerable effect on ventricular pressure and systolic Ca2+ dynamics. Interestingly, blockade of muscarinic receptors by atropine prolonged the duration of phase 2 indicating that, in isolated hearts, there is an intrinsic release of acetylcholine. In addition, the acceleration of repolarization induced by carbachol was blocked by the acetylcholine-mediated K+ current inhibition. Our results reveal the transmural ramifications of autonomic regulation in intact mice hearts and support our hypothesis that Ca2+ influx that triggers ECC occurs in AP phase 1 and not in phase 2.

Altered K+ current profiles underlie cardiac action potential shortening in hyperkalemia and β-adrenergic stimulation.

Hyperkalemia is known to develop in various conditions including vigorous physical exercise. In the heart, hyperkalemia is associated with action potential (AP) shortening that was attributed to altered gating of K+ channels. However, it remains unknown how hyperkalemia changes the profiles of each K+ current under a cardiac AP. Therefore, we recorded the major K+ currents (inward rectifier K+ current, IK1; rapid and slow delayed rectifier K+ currents, IKr and IKs, respectively) using AP-clamp in rabbit ventricular myocytes. As K+ may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these K+ currents. We found that IK1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K+ efflux dominated over the altered channel gating in case of IKr and IKs. Overall, the markedly increased IK1 in hyperkalemia overcame the relative decreases of IKr and IKs during AP, resulting in an increased net repolarizing current during AP phase 3. β-Adrenergic stimulation of IKs also provided further repolarizing power during sympathetic activation, although hyperkalemia limited IKs upregulation. These results indicate that facilitation of IK1 in hyperkalemia and β-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.