Intracellular stimulation and recording from single cardiac cells

Experimental -.. ~~~ Intracellular

Studies Stimulation and Recording

from Single Cardiac Cells* JUNJI USHIYAM.4,

D.D.s., PH.D.t and

Brooklyn,

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(CHANDLER

McC.

BROOKS,PH.D.

New York

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METHODS have been employed to stimulate and record from single cardiac muscie and Trautwein” utilized fibers. Weidmann’ two intracellular microelectrodes, stimulating through one and recording transmembrane potential through another inserted in the same fiber and at a short distance from the first microelectrode. Hoffman3 used a doublebarreled microelectrode, one side to pass current and the other to record transmembrane neurophysiologic studies, a potential. In single intracellular microelectrode often has been used both to stimulate and record.*-6 Because of the rather strong current required to stimulate cardiac fibers, this method has not often been applied to studies of heart muscle. However, it does possess two distinct advantages: Small fibers or cells can be studied, and responses occurring in the immediate vicinity of In our study, the stimulus can be recorded. this latter technic was employed to stimulate and record from single ventricular fibers or Purkinje fibers of dogs.

.l‘he entire process was completed within 15 minutes. Lrntil used, specimens were kept in a warm (37’c.) Tyrode solution which was aerated with a mixture of 95 per cent 0s and 5 per cent COs. The composition of this solution has been described previously.3 During an experiment the preparations were bathed by oxygenated Tyrode solution flowing at a slow rate in a small lucite chamber. The preparations of Purkinje fiber or trabecular muscle were fixed at both ends to the bottom of the bath by a small lucite rod which exerted slight pressure on the tissue. Two silver wires, insulated with enamel except at their cut ends, were attached to one of the lucite rods and served as surface-stimulating elecThe interpolar distance of these electrodes trodes. The temperature of the solution was 2 millimeters. in the bath was kept at 37”~. and monitored by a Cambridge skin temperature meter throughout the experiment. The microelectrodes were made by hand. Class tubing was drawn in a small gas flame and filled with Elec3 molar KC1 solution by the alcohol method.’ trodes of rather low resistance (4 to 8 megohms in Ringer’s solution) were selected for these experiments: since we anticipated passing a relatively large current However, the tips of these electrodes through them. were submicroscopic when examined by a water immersion microscope having a 4,800 magnification \vith blue transmitting light. I‘hp threshold of a single cardiuc cell was determined by passing current between the intracellular microelectrode and an extracellular electrode. Current strength was measured across a 500K resistor in series with the intracellular electrode (Fig. 1A). To minimize the stimulus artifact, a bridge circuit similar to that previously used in testing the excitability of the spinal motor neuron was employed.” Our lorc~ resistance microelectrodes were ohmic when the tips were immersed in l‘yrode solution and tested

METHODS ‘frabecular muscles and Purkinje fibers were obtained from the ventricles of dogs. The operative procedure was as follows. The dogs were anesthetized by an intravenous injection of pentobarbital (40 mg.,/kg.). ‘The entire heart was removed from the body through a midsternal incision and washed with warm Tyrode solution. The ventricles were opened and trabecular muscles and Purkinje fibers were then removed; a small portion of ventricular During this procewall was left adherent to each. dure the heart was immersed in warm Tyrode solution.

* From the Department of Physiology, State University of New York, Downstate Medical Center, Brooklyn, New This study was supported by a grant from the Life Insurance Medical Research Fund. York. t Present address: Kobe Medical College: Kobe, Japan. 688

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FIG. 1. A, simplified diagram of preparation. E = the surface electrodes; P = the circuit to compensate resting potential; S = switch to change the polarity of stimulating current; Relay = magnetic relay operated by square pulses. B, circuit diagram of preamplifier. However, when with currents up to lop6 amperes. the tip touched tissue or penetrated a cell, the resistance became slightly higher at low current strengths; when the current intensity was increased, the electrode resistance also increased irregularly. This change in the apparent resistivity of the electrode mad<* it difficult to balance out the artifact of stimulation by the bridge circuit. Also, the stimulating current. \vhen applied through the 30 megohm resistor (Fig. l-4). had a tendency to escape to the ground To overthrough the grid of the cathode follower. come this. we constructed a special cathode-followerThis amplifier amplifier used in these experiments. was designed to have unity gain over a large range of input signals and consequently approached infinite input impedance (Fig. 1B). ?%r rrctonplar stimulatiq ,tdses which were applied to the bridge circuit were obtained from a small battery (109.5 v.) interrupted by high speed magnetic The intensity of the pulses was controlled by relay. two decade boxes (R, and RJ shown in Figure 1. Square pulses obtained from a Tektronix type 161 pulse generator were applied to the surface electrodes (E in Fig. 1A) through a Grass Stimulus Isolation Unit and were used for extracellular stimulation. NOVEMBER

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FIG. 2. Purkinje fiber responses to direct stimulation. Upper trace shows pulse duration and amplitude. Lower trace shows transmembrane action potentials. A, threshold stimulation; B and C, suprathreshold stimuli. Time markers at 50 msec. intervals; current calibration for stimulus pulses is IO-7 amperes.

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FIG. 3. Trabecular muscle fiber response to direct stimulation. A, subthreshold pulse; B, threshold excitation; C, superimposed threshold and subthreshold responses. It is thought that deflections caused by subthreshold stimuli were largely artifactual. Arrows in B and C show beginning of action potential. Time marks at intervals of 50 msec.: current calibration for stimulus = 10-C amperes.

RESULTS Purkinje Fiber: The records shown in Figure 2 were obtained from a single Purkinje fiber. Current pulses of long duration were passed through an intracellular microelectrode, and resulting changes in transmembrane potential were recorded on the lower trace. The record on the upper trace of each pair shows the duration and intensity of the stimulating current. At the threshold (Fig. 2A) a latency of over 100 milliseconds between the beginning of the stimulus and the upstroke of the transmembrane

FIG. 4 Stimulation of trabecular muscle. Threshold intensities of pulses of various durations are shown. Arrows indicate threshold intensities; time markers at intervals of 50 msec.; current calibration = 10-e amperes.

action potential can be seen. Stronger stimuli (Fig. 2B,C) greatly reduce this latency; however, even when the strength was increased to 10 times the rheobasic requirement, in only 2 of 12 preparations was the latency of response reduced to values much shorter than that shown in Figure 2C. The threshold of the Purkinje THE

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FIG. 5. Break excitation of trabecular muscle during terminal phases of its action potential. Upper tracings show pulse duration and position with respect to action potential shown in lower tracing. Arrows show amplitude of pulse; time marks ( ) at intervals of 100 msec. for B through E.

fiber shown in Figure 2 was 3 X 10-8 amperes; this value was representative of all preparations. Trahmtlar Muscle Fiber: When single fibers of trabecular muscle were stimulated through an intracellular microelectrode, the results obtained differed in two respects from those already described. The threshold for excitation was much higher in muscle fibers, and the latency of responses was much shorter. In the NOVF.MBER

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experiment shown in Figure 3 the threshold for a long duration current pulse (Fig. 3B) was 7 X 10-T amperes; this value is more than 20 times higher than the average threshold of Purkinje fibers. At the sweep speed used for Figure 3 it is impossible to measure the latency of the response; nevertheless, it clearly is much shorter than that obtained for Purkinje fibers. The threshold intensity for pulses of different durations is shown in Figure 4. There was no appreciable lowering of threshold when the stimulus duration was greater than 10 milliseconds. Short negative tests pulses (inward current through the cell membrane) were applied through an intracellular electrode at .various times during the action potential of a trabecular muscle fiber. At a critical time during the early part of the final phase of repolarization (phase 3), break excitation appeared (Fig. 5). The secondary action potentials elicited at the end of the current pulse could be eliminated if the current strength was increased. This result is similar to that reported by Weidmann;s however, the secondary action potentials shown in Figure 5 appeared in an all-or-none manner and clearly differ from the graded depolarization observed previously.8 The duration of the current pulse was not critically related to production of break excitation (Fig. 5); as long as the break of the pulse occurred during the proper phase of repolarization, a secondary action potential was elicited. In contrast, if a pulse of fixed duration was moved earlier or later in the action potential, break excitation was absent regardless of the strength of the stimulus. Safety Factor of Cardiac Muscle Fiber: In some previous work9 we attempted to determine the safety factor in transmission. Such determinations require measurement of the height of the action potential and the threshold current requirement. The method of stimulating and recording from a microelectrode insertecl intracellularly permitted us to obtain an absolute measure of the current required to excite a single cell and the amplitude of the transmembrane action potential. Therefore, it was possible to calculate the safety factor of a single cardiac muscle fiber according to the following relationship : .4n 7-b ,46 X Tn = Safety Factor

An and Ab are the amplitude of the normal action potential and the action potential re-

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6. Elects of a depressant (urethane) on the propagated action potential and threshold of a cardiac muscle fiber. These records were used in calculating safety factor; see text for description. Time marks at intervals of 100 msec. : current calibrations = 10-c amperes. FIG.

corded at the time of block: ‘r?l and ?‘h are the corresponding threshold currents. The records in Figure 6 show an experiment in which the safety factor of muscle was deterThe first action potential of each trace mined. was initiated by stimuli applied at one end of the preparation through extracellular electrodes (E in Fig. 1A) and propagated to the intracellular recording electrode. The second action potential \cas caused by a threshold current

and Brooks passed through the intracellular electrode. ,4s in previous figures, current strength and duration are shown on the upper trace of each pair. After the control records had been obtained (Fig. 6A), the preparation was exposed to Tyrode solution containing 2.5 per cent urethane. There was a progressive reduction in the amplitude of the action potential and a progressive increase in the threshold (Fig. 6B,C). The disappearance of the initial rapid depolarization phase of the action potential under the action of urethane (Fig. 6C,D) may indicate that propagation failed at progressively greatel distances from the intracellular electrode.g By relating the height of the action potential in K (Fig. 6), to the threshold in B, it was found that the safety factor was 1.92. Recovery of the muscle after returning to perfusion with Tyrode solution is shown in Figure 6E; it is apparent that recovery of this preparation was incomplete. The records in Figure 7 were obtained from a similar experiment on Purkinje fibers. In this case the safety factor was found to IW 4. Summation of a local response with thr stimulus delivered through the microelectrade elicited an action potential, as can be SCCII in Figure 7D and E, although stimulation xvitlr the same current (Fig. 7C) at another time failed to produce such a response. The record in Figure 7F was obtained after perfusion with normal Tyrode solution. Although the action potential returned to the control configuration, the threshold current requirement remained increased. In all experiments, after application of urethane there was a gradual increase in threshold .4t up to the time of failure of propagation. this instant there was a marked increase in threshold of both muscle and Purkinje fibers. It could not be determined whether the fibers were excitable at this time because of the limited current which could be passed through the intracellular microelectrode. DISCUSSION The results shown in each figure were confirmed by more than three complete experiments for each sample. Although a large number of experiments of each type were carried out, in many instances there was an abrupt rise in threshold of the cell after application of a strong current; this necessarily terThe inminated that particular experiment. crease observed in threshold may have resulted from damage to the membrane by the stimulus THE AMI’.RIcAX JOURNAI.

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current.

Although propagated action potentials recorded from the same site did not show any changes detectable at the sweep speeds employed, these action potentials may have originated in adjacent parts of the fiber membrane. The observation that thresholds of Purkinje fibers were lower than those of cardiac muscle may have resulted in part from a summation of the applied current with the normal intrinsic excitatory process (slow diastolic depolarization) of the fibers. The time required for such summation may explain the longer latency of response of Purkinje fibers to threshold stimuli. It is unlikely that differences in histologic structure or electrical properties alone could account for the differences in threshold and latency. The stimulating efficacy of the propagated action potential in exciting adjacent regions of cardiac fibers has been found similar to that of pulses of 10 milliseconds’ duration applied during absolute and relative refractoriness.‘O The similarity of the effective duration of the action potential and the utilization time of a rectangular pulse implies that the initial brief overshoot and the late part of the plateau of the transmembrane action potential do not contribute much to propagation. The methods used in these experiments give some evidence concerning the nature of the much discussed “dip” in the recovery of excitability of cardiac muscle.“J2 It was possible to excite a single muscle fiber just at the end of the absolute refractory period or plateau of the action potential by the break of a negative pulse applied through an intracellular microelectrode; however, the same current strength was ineffective in causing break excitation if applied earlier or later in the recovery phase of the action potential. Later, during repolarization, much stronger currents were required. If our interpretation of the “break” excitation at the end of the action potential is correct, then the “dip” is the most plausible point at which spontaneous re-excitation takes place, for under such a condition the minimal gradientn becomes much lower. Fibrillation might be explained on this basis, although there is no actual measurement of minimal gradient for fibrillating muscles. Determination of the safety factor by the intracellular recording method described conceivably would give a somewhat lower value than that obtained by surface recordings from a bundle of fibers. Since propagation in only one fiber is monitored, the presence of other NOVEMBER 1962

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FIG. 7. Effects of urethane on propagated action potentials and threshold current of a Purkinje fiber. Experimental plan as in Figure 6. Urethane applied between A and B. C, local response and failure of intracellular stimulus. D and E, when intracellular stimulus summated with local response, an action potential was evoked. F, recovery in Tyrode solution. Time marks at intervals of 100 msec.; current calibrations = 10-s amperes.

fibers in the bundle would have no effect, while the safety factor for conduction in a bundle would be that of the units possessing the highest safety factor values.

SUMMARY .4 method was devised for stimulating and recording from single cardiac cells using a single intracellular microelectrode and bridge circuit. Purkinje fibers were found to have a much lower threshold but a greater latency of response than trabecular muscle fibers of the same dog heart. Threshold intensities for rectangular pulses ot different duration were determined for muscle, and it was found that prolonging pulse duration he!-ond 10 milliseconds did not permit furthcl lowering of intensity. ,4pplication of brief pulses of inward current flo~v during the terminal phases of trabecular muscle action potential revealed a period, shortb- after the plateau phase, during which break of the current resulted in excitation. Earlier or later application of lhe pulse had no effect. _\ technic was used to determine the safct! factor for conduction in cardiac tissue. The safet\. factor was found to have a \~~luc of apmuscle and 4 in proximately 2 in trabecular Purkinje fibers.

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REI:ERENCES 1.

2.

S. The cffrct of the cardiac membrane potential on the rapid availability of the sodium carrying system. J. Physiol., 127: 213. 1955. TRAUTWEIN, 1% Physiologic> der HrrTirrcsqrrlariWEIDMANN,

13.

tatcn. In: Khythmusstorungcn des Hrrzrns, p. 46. Edited by Sprang. K. Stuttgart. 1957. George Thieme Verlag. HOFFMAN, B. F., KAO, C. Y. and SUCKLING, E. L. Refractoriness in cardiac muscle. Am. J. Physiol, 190: 473, 1957. ARAW, T. and OTANI, T. Response of single motonrurons to direct stimulation in toad’s spinal cord. J. ATmPurophysiol.,18: 472, 1955. FRANK, K. and FUORTES, M. G. F. Stimulation of spinal motoneurons with intracellular electrodes. J. Phy.riol., 134: 451, 1961. USI~IYAM.A,.J., KOIZUMI, K. and BROOKS, C. McC. Excitability of spinal neurons and changes resultin,q from r&c&r formation stimulation. Am. J. i%ysiol., 198: 133, 1960. ‘I‘ASAKI, I.. POLLEY, L,. H. and (>RREGo. F. .\ction potentials from individual elements in cat grniculate and striate cortex. .I. :Veurophysiol., 17: 144, 1054. I1A‘rct of currrnt flow on the memWEIDMANN, s. brane potential of cardiac muscle. J. Physiol., 115: 227. 1951. USHIYAMA, .J. and BROOKS. (i. McC. ‘l‘he safety factor for conduction on cardiac muscles. In preparation. HOFPMAN,B. 1:. Elcctrophysiology ofsingle cardiac ~11s. Hull. .l:eru lhrk Acnd. .Wrd., 35: 689, 1956. BROOKS. C:. h,fcC., HOFFMAN,B. F., SUCKLING, 1,;. I;. and ORIAS. 0. Excitability of the Heart, pp. 119, 141. New York, 1955. Prune and Stratton. HOFPMAN, B. 1:. and (:RANE~IELU, 1’. F. Elcctrophysiology of the Heart, p. 222. New York, 1960. McGraw-Hill Book Co., Inc. ~~XIIYAM.~, J. and BROOKS, C. McC. Hypothermia and minimal gradient requirements for excitation of cardiac musclts. .tnl. _I. Physiol.. 300: 718, 1961.

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