Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres

Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.     

Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery

The electrical responses of the smooth muscle cells of the rabbit common carotid artery to extracellular stimulation were studied in isotonic and hypertonic solution (1.7 times normal tonicity) with microelectrodes. No spontaneous electrical or mechanical activity was recorded when the tissue was in either isotonic or hypertonic solution. The voltage-current relation of smooth muscle cells in the common carotid artery showed marked rectification in both isotonic and hypertonic solutions. In isotonic and hypertonic solutions mean values for membrane potentials were -44.5 and -51.5 mv, for space constants 1.13 and 1.21 mm, and for time constants 212.2 and 238.2 msec, respectively. Addition of 34.3 mM TEA to the solutions caused spontaneous action potentials in the common carotid artery. The action potentials recorded simultaneously from two microelectrodes showed good synchronization. It was concluded that there is electrical transmission between cells of this artery.     

Physiological implications of K accumulation in heart muscle

K+-selective microelectrodes in conjugation with the voltage clamp technique were used to examine the voltage and time dependence of K+ efflux and accumulation in cardiac muscle. K+ efflux per action potential is about 10 to 30 pmoles/cm2 per sec. Accumulation of K+ in the paracellular space plays an important role in regulation of action potential duration, so that the [K+]o prior to generation of an action potential determines the duration of following action potential. This regulation is brought about by the shift of inward rectifying K+ current along the voltage axis, so at higher [K+]o there is more outward current at plateau potentials. Monitoring [K+]o after a period of rapid beating provides quantitative data regarding Na-pump activity. The data suggest the Na-pump is electrogenic, making it difficult to assess the extent of K+ accumulation from the measurements of resting potential alone. These studies indicate that changes in [K+]o not only reflect outward membrane currents and Na-pump activity, but also play an important physiological regulatory role in determining the duration of the action potential.     

Electrophysiological analysis of the effect of cortisone on the rat neuromuscular apparatus]

The influence of cortisone (1.5 mg per 100 g of body weight, daily, for 10 days) on the neuro-muscular system was studied in rats in situ. The action potentials of the nerve and muscle were recorded with the extracellular electrodes. The rest potentials (RP) of the muscle fibers and the miniature end-plate potentials (MEPP) were recorded with the intracellular glass microelectrodes. A decrease of the RP and the MEPP amplitude, and an increase of the MEPP frequency and prolongation of the neuromuscular transmission time were revealed in rats given daily doses of cortisone, 1.5 mg/100 g of body weight, during 10 days; reliability of the neuro-muscular transmission (acceleration of the fall of the muscle action potential amplitude during tetanus) proved to decrease under the action of cortisone.     

Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat

Small pulmonary arteries (less than 300 micron) from cats were mounted in myographs to record mechanical and electrical responses to hypoxia. When these preparations were exposed to a PO2 of 30-50 Torr after equilibration at 300 Torr they consistently developed active force, which increased or decreased in amplitude as [Ca2+] was raised or lowered, respectively, and was blocked on addition of verapamil. Intracellular electrical recording with glass microelectrodes demonstrated membrane depolarization and action potential generation when PO2 was lowered. Steady-state voltage vs. applied current curves obtained before and during hypoxia showed a significant reduction in input resistance. The relationship between membrane potential and extracellular K+ was not different during hypoxia compared with control, suggesting that there were not marked changes in K+ permeability under this condition. In the presence of verapamil to block Ca2+ inward current the hypoxia-induced action potentials were abolished concomitant with partial membrane repolarization. The results of these studies suggest that in certain isolated pulmonary arteries hypoxia induces contraction by a mechanism involving an increased Ca2+ conductance. These data suggest that the sensor involved in hypoxic pulmonary vasoconstriction may lie within the vessel wall and somehow mediates changes in smooth muscle ionic conductances.     

Effect of TaiCatoxin (TCX) on the electrophysiological,mechanical and biochemical characteristics of spontaneously beating ventricular cardiomyocytes

TaiCatoxin (TCX), a complex toxin isolated from Taipan snake venom, is believed to have a specific blocking activity on voltage-dependent cardiac calcium channels. The aim of this study was to investigate the effects of TCX on a broad range of heart muscle cell functions, i.e. electrophysiology, contractility, automaticity and the related biochemical modifications. Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded with glass microelectrodes. The contractions were monitored photometrically. TCX decreased the action potential amplitudes, mainly by lowering the plateau. The action potential duration and the contraction parameters were decreased. Although TCX has a minor overall negative chronotropic effect, it evoked transient but severe arrhythmias and prolonged changes in the intercellular electrical coupling. Moreover, the action of TCX appeared to be dose-dependent. These effects are consistent with a specific blockade of the L-type, voltage-dependent calcium channels, but effects of other components of the toxin complex cannot be excluded. TCX also exhibits phospholipase A₂ activity leading to the release of lysophospholipids and FFA (acyl CoA and acyl carnitine), which have detrimental effects on cellular integrity and function.     

Disorder of electromechanical coupling in the cells of the myocardium in protracted crush syndrome

Experiments on papillary muscles of normal (control) rabbits and of those with the compression syndrome (CS) were made to explore the action of the control and "syndromic" blood plasma on electric and contractile activity of the myocardium. Isometric contractions of myocardial preparations were recorded at varying stimulation frequencies (0.1-2 Hz). Intracellular rest potentials (RP) and action potentials (AP) were led away with the aid of glass microelectrodes filled with 2.5 M KCl. The replacement of Tyrode solution by the control plasma raised the amplitude of papillary muscle contractions, that being greater as regards the muscles from rabbits with the CS. The "syndromic" plasma (diluted by Tyrode solution in a 1:1 ratio) markedly inhibited the amplitude of contractions of papillary muscles from both the control rabbits and animals with the CS. Reduction of the contractions induced by the "syndromic" plasma seen in all the preparations was followed by two patterns of changes in electrical activity of myocardial fibers. In one pattern, the RP, the amplitude and duration of the AP declined. In the other, on the contrary, the changes were reduced to a greater AP duration. The conclusion is made about the absence of a direct relationship between the decrease in myocardial contractility and changes in intracellular potentials induced by the "syndromic" plasma. It is suggested that the "syndromic" plasma deranges the process of stimulation and contraction coupling in heart papillary muscles.     

Simple techniques suitable for student use to record action potentials from the frog heart

Demonstrating action potentials during class experiments is very educational for science students. It is not easy, however, to obtain a stable intracellular recording of action potentials from the conventionally used skeletal muscle cells, because the tip of a glass microelectrode often comes out or breaks due to muscle contraction. Here, I present a much simpler recording method using a flexible polyethylene electrode with a wide orifice (approximately 1 mm) for a bullfrog heart beating on automaticity. Extracellular recordings of action potentials (electrocardiogram) can be obtained by placing an electrode on the cardiac surface, and transmembrane potentials can be obtained by rupturing the membrane with negative pressure, i.e., whole cell configuration. Once attached to the heart by suction, the polyethylene electrode does not easily come off during contraction of the heart. Perfusion of the heart via the postcaval vein offers us opportunities for observing the effects of either changing ionic compositions of solutions or applying drugs. The techniques shown here provide a simple and convenient way to perform a variety of class experiments.     

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.     

Use of micromachined probes for the recording of cardiac electrograms in isolated heart tissues

Micromachined probes, with iridium (Ir) microelectrodes on silicon shanks, were evaluated to assess their suitability for cardiac electrogram recording. The electrochemical activation (anodic oxidation) procedure for the circular Ir microelectrode was investigated using the square wave potential according to the electrode size, number of cycles, and cathodic-anodic potential level of the square wave. Increase in the charge storage capacity was pronounced either in smaller electrodes or with higher potential level of the square wave. The electrode impedance reduced in a similar manner with increasing number of cycle irrespective of the electrode size. With either lower potential level (-0.70/+0.60 V) or smaller number of cycle (200 cycles) than those for the activation of stimulating electrode, the likelihood of overactivation of the recording microelectrode can be minimized. These anodic IrOx film (AIROF) microelectrodes were used for the recording of extracellular electrograms in two different ex vivo cardiac tissue preparations. A single-shank microprobe was applied to the left ventricle of a mouse heart. Both the spontaneous and paced transmural responses propagating between epicardium and endocardium were obtained. Longitudinal cardiac wavefronts propagating along the rabbit papillary muscle were also recorded with a unique multiple-shank design. The measured mean amplitude and the propagation velocity of the extracellular voltage were 12.2 +/- 1.8 mV and 58.9 +/- 2.2 cm/s, respectively (n = 27). These microprobes with precisely defined electrode spacing make a useful tool for the spatial and temporal mapping of electrical properties in isolated heart tissues ex vivo.     

Anti-arrhythmia action of the antioxidant SD-6 in the development of ischemic and reperfusion rhythm disorders of the isolated rat heart

An antiarrhythmic action of water-soluble antioxidant SD-6 from 3-hydroxypyridine class and its effect on the transmembrane potentials were studied using the isolated rat heart and papillary muscle. Ischemia was induced by the occlusion of the left anterior descending coronary artery. 10 minutes later the ligation was removed and reperfusion was achieved. In the control, ischemia induced premature ventricular complexes, tachycardia and, in some cases, fibrillation. During perfusion total fibrillation occurred in 100% of the experiments. SD-6 in the doses of 10(-6) g/ml and 5 X 10(-6) g/ml significantly reduced the incidence of fibrillation and tachycardia. In the experiments on the papillary muscle SD-6 during reperfusion completely normalized the action potential duration and removed depolarization developed in hypoxia, which suggests the ability of the antioxidant to block reperfusion-induced arrhythmias by normalization of the parameters of electrical heterogeneity. These data show that the origin of reperfusion-induced arrhythmias is connected with the activation of free radical metabolites and that their scavengers--synthetic antioxidants from 3-hydroxypyridine class--can be used as new antiarrhythmic agents.     

Problems of recordings of nervous activity with glass microelectrodes in the CNS of insects

1. Extracellular single unit recordings with glass microelectrodes in the central nervous system of insects display action potentials of variable amplitude, polarity and time course. This phenomenon is due to capacitive influences at the electrode in contact with the tissue. This is demonstrated by an electrical model circuit simulating extracellular recording conditions. 2. Extracellularly recorded potentials often are very similar to intracellularly recorded ones. Criteria for the decision whether the electrode is intracellularly or not are discussed. 3. Action potentials and slow potentials were recorded simultaneously in the acoustic neuropiles of Locusta. Since slow potentials may not only be distorted by capacitive properties of both the tissue and the electrode, but also are influenced by the anatomical organization of the nervous tissue, their interpretation is ambiguous.     

An optical monitor of tension for small cardiac preparations.

When a light beam is focused on a muscle preparation which is allowed to contract, large changes in the intensity of the emerging light accompany the contraction. These movement-related optical signals were studied and compared to simultaneous measurements of force in isolated cardiac Purkinje fibers. The two signals were compared in response to action potentials and to graded changes in membrane potential controlled under voltage clamp. These experiments indicate that the optical signal is a sensitive monitor of tension development under these conditions. This technique is particularly well-suited to force measurements in smaller preparations in which direct mechanical techniques are not feasible.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Active electric properties of cardiac muscle

A model of electrical activity in the heart has been developed that treats the intracellular domain and the extracellular domain as electrical syncytia with anisotropic resistivities (bi-syncytial model). At the microscopic level, propagation is assumed to proceed primarily along the axes of individual cells. Considerations at the macroscopic level relate the transmembrane current to the intracellular and extracellular resistivity and the transmembrane potential. The result is a relationship between instantaneous extracellular potentials and cardiac action potentials.     

A voltage clamp study of the sodium, calcium and chloride spikes of chick skeletal muscle cells grown in tissue culture.

Conventional voltage clamp techniques with microelectrodes were applied to chick muscle cells grown in tissue culture. The similarities and differences in electrophysiological data obtained from normal myotubular muscle fibers and from rounded myosacs, produced by incubation with colchicine, were examined. Under voltage clamp both cellular types generated three distinguishable voltage and time-dependent currents which corresponded, respectively, to the Na⁺, Ca²⁺, and Cl^? spikes evoked under constant current conditions. The presumed Ca²⁺ currents were too small to allow quantitative comparisons. In myosacs, but not in myotubes, there was good correspondence, for both the Na⁺ and Cl^? systems, between their spike thresholds and peak membrane potentials, measured under constant current conditions, and their current thresholds and reversal potentials, measured under voltage clamp conditions. This correspondence is attributed to the isopotentiality of the myosac intracellular space and suggests that myosacs provide more accurate quantitative data in voltage clamp studies than myotubes.     

An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

On the electrotonic spread in cardiac muscle of the mouse

As an appropriate model which can simulate the cardiac working muscle with respect to the passive electrical spread, a lattice whose sides have linear cable properties is presented, and the passive potential spread on the model is mathematically analyzed in the fiber direction. Distribution of electrotonic potential in the fiber direction was measured with a pair of intracellular microelectrodes in the cardiac muscle fiber of mouse. By employing “pencil type” microelectrodes potential distribution in the transverse direction within a fiber was also measured. This transverse effect was differentiated from the longitudinal potential distribution. A tonically applied potential at any point of a cell interior spreads continuously in a manner described by a Bessel function. Using appropriate electrical and morphological parameters the experimental results proved to fit the curve obtained from numerical calculation on the model. The apparent length constant obtained for smaller distances (less than 100 μ) from the current source was 70 μ, and it increases as the distance becomes larger. At a point inside the fiber the resistance to the extracellular fluid ranged from 200 to 600 KΩ. The influence of coupling resistance between current and recording electrodes on the measurement of electrotonic potential was examined for small interelectrode distance.     

Cesium blockade of delayed outward currents and electrically induced pacemaker activity in mammalian ventricular myocardium

The effects of Cs+, 5-25 mM, were studied in cat and guinea pig papillary muscles using voltage clamp and current clamp techniques. In solutions containing normal K+, the major effects of Cs+ were depolarization of the resting potential and reduction of the delayed outward current (ixl) between -80 and -20 mV. Both inward and outward portions of the isochronal current voltage relation (l-s clamps) were reduced by extracellular Cs+. This resulted in a substantial reduction of inward rectification and, by subtraction from the normal I-V relationship, the definition of a Cs+-sensitive component of current. Under current clamp conditions, 5-10 mM Cs+ produced a dose-dependent slowing of repetitive firing induced by depolarization. At higher concentrations (25 mM) the resting potential was depolarized and repetitive activity could not be induced by further depolarization. However, release of hyperpolarizing pulses was followed by prolonged bursts of repetitive action potentials, suggesting partial reversal of blockade or participation of another pacemaker process. The experimental results and a numerical simulation show that under readily attainable conditions, reduction in an outward pacemaker current may slow pacemaker activity.     

The course of ionic transmembrane currents during cardiac action potentials.

The course of the total transmembrane ionic current (Ii) during a natural action potential (AP) was reconstructed from a family of current traces recorded for single voltage clamp depolarization steps to various levels. The experiments were performed on 9 papillary cat muscles driven at 0.5 per second in oxygenated 31 degrees C Tyrode. Under varying experimental conditions very good agreement was found between the resulting Ii curve and another indicator of Ii, the first time derivative of the AP (dV/dt). Furthermore, the coefficient needed to adjust dV/dt to reconstructed Ii may serve as an indicator of the membrane capacity. The results suggest the validity of the employed approximation and, in general, the adequacy of the sucrose gap technique applied to cardiac muscle.