Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.     

3D current-voltage-time surfaces unveil critical repolarization differences underlying similar cardiac action potentials: A model study

The number of mathematical models of cardiac cellular excitability is rapidly growing, and compact graphical representations of their properties can make new acquisitions available for a broader range of scientists in cardiac field. Particularly, the intrinsic over-determination of the model equations systems when fitted only to action potential (AP) waveform and the fact that they are frequently tuned on data covering only a relatively narrow range of dynamic conditions, often lead modellers to compare very similar AP profiles, which underlie though quite different excitable properties. In this study I discuss a novel compact 3D representation of the cardiac cellular AP, where the third dimension represents the instantaneous current–voltage profile of the membrane, measured as repolarization proceeds. Measurements of this type have been used previously for in vivo experiments, and are adopted here iteratively at a very high time, voltage, current-resolution on (i) the same human ventricular model, endowed with two different parameters sets which generate the same AP waveform, and on (ii) three different models of the same human ventricular cell type. In these 3D representations, the AP waveforms lie at the intersection between instantaneous time–voltage–current surfaces and the zero-current plane. Different surfaces can share the same intersection and therefore the same AP; in these cases, the morphology of the current surface provides a compact view of important differences within corresponding repolarization dynamics.Refractory period, supernormal excitability window, and extent of repolarization reserve can be visualized at once. Two pivotal dynamical properties can be precisely assessed, i.e. all-or-nothing repolarization window and membrane resistance during recovery. I discuss differences in these properties among the membranes under study, and show relevant implications for cardiac cellular repolarization.     

Calcium-activated conductance in skate electroreceptors: current clamp experiments

When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa.     

Recovery of action potentials and twitches after K-contractures in frog skeletal muscle

To give information about intracellular Ca2+ translocation during and after K-contractures in vertebrate skeletal muscle fibers, we examined recovery of action potentials and twitches after interruption and spontaneous relaxation of K-contractures at low temperature (3 degrees C) that greatly reduced the rate of Ca2+ reuptake by the sarcoplasmic reticulum. On membrane repolarization interrupting K-contractures, the amplitude of both action potentials and twitches recovered quickly, while the falling phase of action potential was markedly slowed at first to prolong its refractory period, so that repetitive stimulation (20 Hz) did not produce a complete tetanus. Meanwhile, on membrane repolarization after spontaneous relaxation of K-contractures, the action potentials were markedly reduced in amplitude and prolonged in duration at first, also resulting in prolonged refractory period. These results are discussed in connection with Ca2+ absorption to the surface and transverse tubule membranes, producing changes in action potential kinetics.     

Membrane Currents in Mammalian Ventricular Heart Muscle Fibers Using a Voltage-Clamp Technique

Bundles of sheep ventricular fibers were voltage-clamped utilizing a modified sucrose gap technique and intracellular voltage control. An action potential was fired off in the usual way, and the clamp circuit was switched on at preselected times during activity. Clamping the membrane back to its resting potential during the early part of an action potential resulted in a surge of inward current. The initial amplitude of this current surge decreased as the clamp was switched on progressively later during the action potential. Inward current decreasing as a function of time was also recorded if the membrane potential was clamped beyond the presumed K equilibrium potential (to -130 mv). Clamping the membrane to the inside positive range (+40 mv to +60 mv) at different times of an action potential resulted in a step of outward current which was not time-dependent. The results suggest that normal repolarization of sheep ventricle depends on a time-dependent decrease of inward current (Na, Ca) rather than on a time-dependent increase of outward current (K).     

Effect of diclofop on the membrane potentials of herbicide-resistant and -susceptible annual ryegrass root tips

Electrophysiological measurements were made on root tip cells in the elongation zone of diclofop-methyl-resistant (SR4/84) and -susceptible (SRS2) biotypes of annual ryegrass (Lolium rigidum Gaud.) from Australia. The phytotoxic action of diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate) on susceptible whole plants was completely reversed by a simultaneous application of 2,4-dichlorophenoxyacetic acid (dimethylamine salt). The phytotoxic acid metabolite, diclofop (50 micromolar), depolarized membrane potentials of both biotypes to a steady-state level within 10 to 15 minutes. Repolarization of the membrane potential occurred only in the resistant biotype following removal of diclofop. The resistant biotype has an intrinsic ability to reestablish the electrogenic membrane potential, whereas the susceptible biotype required an exogeneous source of IAA to induce partial repolarization. Both biotypes were susceptible to depolarization by carbonylcyanide-m-chlorophenylhy-drazone (CCCP), and their membrane potentials recovered upon removal of CCCP. A 15-minute pretreatment with p-chloromercuribenzenesulphonic acid (PCMBS) blocked the depolarizing action of diclofop in both biotypes. However, PCMBS had no effect on the activity of CCCP. The action of diclofop appears to involve a site-specific interaction at the plasmalemma in both Lolium biotypes to cause the increased influx of protons into sensitive cells. The differential response of membrane depolarization and repolarization to diclofop treatment may be a significant initial reaction in the eventual phytotoxic action of the herbicide.     

The sites for mechano-electric conversion in a Pacinian corpuscle

The sensory nerve ending in the Pacinian corpuscle is surrounded by a non-nervous capsular structure which occupies about 99.9 per cent of the corpuscle's entire mass. After extirpation of practically all of the non-nervous structure, the sense organ's remains continue to function as a mechano-receptor, namely to produce generator and all-or-nothing potentials in response to mechanical stimuli. Compression of the first intracorpuscular node of Ranvier abolishes the production of "all-or-nothing" potentials in the corpuscle. Graded generator potentials constitute then the only response to mechanical stimulation. This reveals that the first node is the site of origin of the all-or-nothing potential and that the non-myelinated ending is incapable of producing all-or-nothing responses in response to mechanical stimulation. Compression of the entire length of non-myelinated ending suppresses the production of generator potentials. Partial compression of the ending abolishes mechano-responsiveness only of the compressed part. The intact remains of the ending continue to give generator potentials upon mechanical stimulation. This suggests that the generator potential arises at functionally independent membrane parts distributed all over the non-myelinated nerve ending. 24 to 36 hours after denervation of the corpuscle by transection of its sensory axon, no sign of electric activity is detected. Failure of mechano-reception at the nerve ending precedes that of conduction at the degenerating myelinated axon.     

Computed Action Potentials for Purkinje Fiber Membranes with Resistance and Capacitance in Series

The Hodgkin-Huxley equations, as modified by Noble for computation of Purkinje fiber action potentials, have been solved numerically for a membrane whose equivalent circuit contains a constant resistance in series with part of the capacitance. The rates of depolarization and repolarization of the computed action potential have thereby been brought into agreement with measured values. Possible explanations of the frequently observed pre-plateau notch and of fibrillatory activity arise. The effects of a time-dependent K conductance dependent on the second power of the parameter n, instead of the fourth, have also been considered.     

Propagated repolarization in heart muscle

The effect of current flow on the transmembrane action potential of single fibers of ventricular muscle has been examined. Pulses of repolarizing current applied during the plateau of the action potential displace membrane potential much more than do pulses of depolarizing current. The application of sufficiently strong pulses of repolarizing current initiates sustained repolarization which persists after the end of the pulse. This sustained repolarization appears to propagate throughout the length of the fiber. Demonstration of propagated repolarization is made difficult by appearance of break excitation at the end of the repolarizing pulse. The thresholds for sustained repolarization and break excitation are separated by reducing the concentration of Ca⁺⁺ in the environment of the fiber. In fibers in such an environment it is easier to demonstrate apparently propagated repolarization and also, by further increase of the strength of the repolarizing current, to demonstrate graded break excitation.     

Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.     

The Repolarization Phase of the Cardiac Ventricular Action Potential: A Time-Dependent System of Membrane Conductances

A system for the generation of the repolarization phase of the ventricular action potential is described. The system is based on time-dependent changes in membrane conductance to sodium and potassium ions. However, the changes in conductance during an action potential retain a degree of voltage dependence through the initial conditions which depend on previous depolarizations of the membrane. The equations describing the system were solved with an analog computer and various action potential forms are reproduced. The effects of hyperpolarizing and depolarizing current applied during an action potential are investigated. The changes in shape of an action potential after a change in the rate of stimulation show partial agreement with previous experimental findings. The applicability of time-dependent and voltage-dependent systems for the generation of the repolarization phase of the ventricular action potential is discussed.     

Phase Plane Trajectories of the Muscle Spike Potential

To facilitate a study of the transmembrane action current, the striated muscle spike potential was recorded against its first time derivative. The specialized recording methods are described, as well as several mathematical transformations between a coordinate system in V, t, and the present coordinate system in V, dV/dt. The particular properties of the present recording method permitted an estimation for the “sodium” potential in muscle fibers at +39 mv. The maximum membrane conductance during spike production is in the order of 150 to 200 mmhos/cm². The changes in the shape of the recorded response indicate that the ionic currents and membrane conductances are reduced by Tris buffer or hypertonic Ringer's fluid. However, no marked changes in the properties of active membrane were observed when chloride ion was replaced by sulfate.     

Effect of 3-5-3'triiodothyronine treatment on cardiac action potential of streptozotocin-induced diabetic rat

Electrical activity of rat atrium of streptozotocin-diabetic and control rats was compared. (i) As occurs in the ventricle, diabetes lengthens the cardiac atrial action potential. (ii) Treatment by T3 of diabetic animals decreases action potential duration to normal values and causes partial recovery in plateau decay during the late phase of repolarization. (iii) T3 treatment however, does not completely normalized the action potential of the diabetic rat atrium, which remains abnormal during the early phase of repolarization. These results demonstrate that some defects in membrane mechanisms involved in the early phase of action potential repolarization are attributable solely to diabetes. The possible nature of these mechanisms is discussed.     

Contributions of HERG K+ current to repolarization of the human ventricular action potential

Action potential repolarization in the mammalian heart is governed by interactions of a number of time- and voltage-dependent channel-mediated currents, as well as contributions from the Na+/Ca2+ exchanger and the Na+/K+ pump. Recent work has shown that one of the K+ currents (HERG) which contributes to repolarization in mammalian ventricle is a locus at which a number of point mutations can have significant functional consequences. In addition, the remarkable sensitivity of this K+ channel isoform to inhibition by a variety of pharmacological agents and clinical drugs has resulted in HERG being a major focus for Safety Pharmacology requirements. For these reasons we and others have attempted to define the functional role for HERG-mediated K+ currents in repolarization of the action potential in the human ventricle. Here, we describe and evaluate changes in the formulations for two K+ currents, IK1 and HERG (or IK,r), within the framework of ten Tusscher model of the human ventricular action potential. In this computational study, new mathematical formulations for the two nonlinear K+ conductances, IK1 and HERG, have been developed based upon experimental data obtained from electrophysiological studies of excised human ventricular tissue and/or myocytes. The resulting mathematical model provides much improved simulations of the relative sizes and time courses of the K+ currents which modulate repolarization. Our new formulation represents an important first step in defining the mechanism(s) of repolarization of the membrane action potential in the human ventricle. Our overall goal is to understand the genesis of the T-wave of the human electrocardiogram.     

Dynamics of the inward rectifier K+ current during the action potential of guinea pig ventricular myocytes.

The potassium selective, inward rectifier current (IK1) is known to be responsible for maintaining the resting membrane potential of quiescent ventricular myocytes. However, the contribution of this current to the different phases of the cardiac action potential has not been adequately established. In the present study, we have used the action potential clamp (APC) technique to characterize the dynamic changes of a cesium-sensitive (i.e., Ik1) current which occur during the action potential. Our results show that (a) Ik1 is present during depolarization, as well as in the final phase of repolarization of the cardiac action potential. (b) The current reaches the zone of inward-going rectification before the regenerative action potential ensues. (c) The maximal outward current amplitude during repolarization is significantly lower than during depolarization, which supports the hypothesis that in adult guinea pig ventricular myocytes, Ik1 rectification is accentuated during the action potential plateau. Our results stress the importance of Ik1 in the modulation of cell excitability in the ventricular myocyte.     

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.     

The Action Potentials and Currents on the I-V Plane in the Molluscan Neuron

The action potentials and the corresponding transmembrane currents, directly recorded in the F1 neuron of Helix aspersa by the Self-clamp Technique, were plotted on the I-V plane to represent the real electrical cycle of the cell membrane during activity. The membrane electrical cycle, experimentally obtained, agreed in several aspects with a similar cycle obtained from calculated data on the giant axon of Loligo, but not for the sign, with the consequence of a different localization, as far as voltage and time are concerned, of the negative impedance period. The negative impedance proved to be −614 ± 181 Ω cm² and corresponded to the late phase of the repolarization after the action potential peak. A constant positive impedance was found of 522 ± 131 Ω cm² during the ascending tract of the action potential. These two results are in contrast with previous analyses. The simultaneous availability of the conjugate voltage and current directly measured signals led to the immediate representation of the membrane total conductance in its real time course during activity, in agreement with the Hodgkin and Huxley predictive model. The peak conductance was 1.9 ± 0.7 mmho/cm² in this preparation. The electrical work spent to sustain a single active event proved to be 70 ± 19 nJ/cm². A vectorial representation of the membrane electrical activity is proposed to describe analytically the characteristic behaviour of excitable cells, as well as a new method that utilizes the only action potential to measure the threshold potential in spontaneously discharging cells. The proposed new experimental protocol, based on the use of the Self-clamp Technique, proved to be faster, easier, more productive when compared with the conventional methods; it could be used advantageously in the electrophysiological studies on excitable cells both to define the basic conditions of the investigated preparation and to directly evaluate the effects of subsequent pharmacological stimulations.     

Contributions of HERG current to repolarization of the human ventricular action potential

Action potential repolarization in the mammalian heart is governed by interactions of a number of time- and voltage-dependent channel-mediated currents, as well as contributions from the Na⁺/Ca²⁺ exchanger and the Na⁺/K⁺ pump. Recent work has shown that one of the K⁺ currents (HERG) which contributes to repolarization in mammalian ventricle is a locus at which a number of point mutations can have significant functional consequences. In addition, the remarkable sensitivity of this K⁺ channel isoform to inhibition by a variety of pharmacological agents and clinical drugs has resulted in HERG being a major focus for Safety Pharmacology requirements.For these reasons we and others have attempted to define the functional role for HERG-mediated K⁺ currents in repolarization of the action potential in the human ventricle. Here, we describe and evaluate changes in the formulations for two K⁺ currents, I_K1 and HERG (or I_K,r), within the framework of ten Tusscher model of the human ventricular action potential. In this computational study, new mathematical formulations for the two nonlinear K⁺ conductances, I_K1 and HERG, have been developed based upon experimental data obtained from electrophysiological studies of excised human ventricular tissue and/or myocytes. The resulting mathematical model provides much improved simulations of the relative sizes and time courses of the K⁺ currents which modulate repolarization. Our new formulation represents an important first step in defining the mechanism(s) of repolarization of the membrane action potential in the human ventricle. Our overall goal is to understand the genesis of the T-wave of the human electrocardiogram.     

The Voltage Dependence of the Cardiac Membrane Conductance

Solutions have been computed for the point polarization of a sheet-like membrane obeying the equations used previously (Noble, 1960, 1962) to reproduce the Purkinje fiber action potential. It was found that, in spite of the gross non-linearity of the membrane current-voltage relations, the relations between total polarizing current and displacement of membrane potential at various distances from the polarizing electrode are remarkably linear. It is therefore concluded that Johnson and Tille's (1960, 1961) results showing linear polarizing current-voltage relations obtained by passing current through the membrane from a microelectrode during the plateau of the rabbit ventricular action potential do not conflict with the Hodgkin-Huxley theory of electrical activity.     

Excitation-contraction coupling in myocardium: implications of calcium release and Na+-Ca2+ exchange

In this paper, we present evidence in support of the hypothesis that electrogenic Na+-Ca2+ exchange is responsible for three phenomena in rat cardiac muscle: the slow repolarization phase of the action potential, the time course of the mechanical recovery process, and the development of triggered arrhythmias. It was shown that the duration of the slow phase of repolarization of the action potential varies in proportion to the Na+ concentration gradient and inversely with the Ca2+ concentration gradient over the cell membrane. This suggested that Na+-Ca2+ exchange can generate a current of sufficient magnitude to maintain the membrane depolarized at a level of -60 mV. The mechanical restitution process of rat cardiac trabeculae was shown to exhibit three phase. The first phase, alpha, probably reflects rapid transport of calcium in the sarcoplasmic reticulum from the uptake sites to the release sites. After the initial increase of force during alpha, force rises further during phase beta and then declines during phase gamma. During all phases, force increases with the extracellular calcium concentration. beta is accelerated by preceding extrasystoles, while an increase of the heart rate causes force to increase at approximately the same rate but to a higher level during phase beta. These observations are compatible with a model in which the sarcoplasmic reticulum sequesters calcium from the cytosol, while the membrane of the sarcoplasmic reticulum is assumed to exhibit also a small leak of calcium into the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)