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Cardiovascular Research 2002 53(1):1-5; doi:10.1016/S0008-6363(01)00502-8
© 2002 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

And then came the microelectrode

Brian F Hoffman*

College of Physicians and Surgeons, Columbia University New York, NY 10032, USA

* Address for correspondence: 600 Gropetree Drive, Apt. 6 EM, Key Biscane, FL 33149, USA

accepted 4 October 2001

It is difficult to fully appreciate the contributions made by the microelectrode to the understanding of cardiac electrophysiology without some idea of the prior state of the field. What was known about cardiac electrophysiology had been derived largely from studies of the electrocardiogram and cardiac electrogram and by the application of stimuli, usually electrical, to the heart. The voltage–time course of the atrial and ventricular action potentials had been revealed by the recording of monophasic potentials through suction electrodes or other means of registering injury potentials. The various phases of refractoriness had been defined by stimulation but this information usually was not related to the monophasic action potential. Evidence of the basic mechanisms underlying the observed phenomena was largely lacking. The major contributions to understanding the nature and mechanisms for arrhythmias and conduction disturbances resulted from precise measurements of the electrocardiogram and careful deductive reasoning. Early studies on in vitro preparations [1] had demonstrated reentrant excitation and identified an inexcitable barrier, unidirectional block and slow conduction as the necessary substrate for this arrhythmia. These abnormalities were attributed to a vaguely described depression of excitability. Some ectopic rhythms were ascribed to abnormal impulse generation and several theories as to mechanism were presented but largely without experimental justification. All in all one knew what was happening in the heart but without an understanding of why.

The ability to record the transmembrane potentials of cardiac muscle brought about a rapid and immense increase in understanding of the basis for normal and abnormal cardiac electrical activity. Intracellular electrodes had been employed to study giant nerve fibers [2] and permitted the brilliant experiments and deductions by Hodgkin, Huxley and Katz [3,4] among others. These experiments provided for the first time an understanding of the conductance changes and ionic currents causing the depolarization and repolarization shown by the nerve action potential. Microelectrodes subsequently were developed and used to record from single skeletal muscle fibers [5]. This technique finally was adapted to studies on cardiac cells. The landmark study was performed by Draper and Weidmann [6]. To avoid the problems caused by cardiac contraction — dislodgement or breakage of the microelectrode — they studied bundles of Purkinje fibers from the hearts of ungulates. These fibers have a large diameter and contract only weakly. They thus were well-suited for impalement with a microelectrode. These experiments revealed the true voltage–time course of the transmembrane action potential, the magnitudes of the resting potential and action potential upstroke, the various phases of repolarization and the role of slow diastolic depolarization in causing automatic firing. The various phases or repolarization were identified as phases 1, 2 and 3 and the interval between the end or repolarization and the next action potential upstroke as phase 4. Subsequently the action potential upstroke was identified as phase 0. The microelectrode method subsequently was adopted by many investigators and used to study a variety of cardiac tissues in an effort to define the electrophysiological basis for their unique electrical activity.

One important early study was directed at recording transmembrane potentials from the pacemaker cells of the sinoatrial node. It was known from unipolar electrogram recordings and measurements of impulse spread that the normal cardiac pacemaker was located in the sinus node but the mechanism for spontaneous impulse generation had not been demonstrated. Through use of the microelectrode, West and Irisawa [7,8] showed that pacemaker activity resulted from slow diastolic depolarization, as in the Purkinje fiber. The basis for this slow depolarization was not fully explained for some years and awaited the application of the patch clamp method. The microelectrode did show that the maximum diastolic potential of sinus node fibers was less negative than in Purkinje or muscle fibers and that the upstroke of the action potential was quite slow. Later it was found that this slow response action potential resulted from an inward calcium current rather than the sodium current causing phase 0 in Purkinje fibers and atrial and ventricular muscle. Control of sinus pacemaker rate by the nervous system also was explained at the observational level. Norepinephrine increased the slope of phase 4 depolarization whereas acetylcholine increased maximum diastolic potential and decreased the slope of phase 4. Obviously, once this mechanism for automatic firing had been described it became possible to look for this characteristic change in transmembrane potential at sites of ectopic impulse generation. This search ultimately led to the description of multiple mechanisms for impulse generation as described subsequently [9].

The spread of the impulse through the atrioventricular node had presented many problems to the early physiologists. It was known that the spread of the impulse from the atria to the ventricles, or from the ventricles to the atria, was delayed in the region of the a–v node. The magnitude of this delay was a function of heart rate or the prematurity of the impulse. Sufficiently premature impulses failed to conduct and with high rates alternate impulses were blocked or there were other ratios of transmission. The electrophysiological basis for these phenomena was unknown. Once again the microelectrode provided new understanding of an old problem. Records obtained from the a–v node of the rabbit heart [10] showed that as the impulse spread from the atria to the His bundle the action potential underwent a series of changes. As the impulse moved from atrial fibers into the node there was a progressive decrease in resting potential and the slope of phase 0 until the action potential resembled that recorded from primary pacemaker fibers of the sinus node. Distal to this site generating slow responses the transmembrane potentials changed in the opposite direction: resting potential and slope of phase 0 increased, as did action potential duration, until signals typical of specialized fibers were recorded from the upper His bundle. Conduction velocity changed in parallel with the action potential, slowing maximally in fibers with the slowest action potential upstroke. The zones of transition in the action potential were designated as A–N, N and N–H. The N zone was the site of block of premature impulses; this was later ascribed to the slow recovery of availability of the calcium current responsible for the slow responses. A major contribution to understanding reentrant a–v nodal rhythms was the demonstration of dual pathways for transmission of impulses through the node [11].

One of the most important contributions that came from studies with the microelectrode was the development of an improved understanding of electrophysiological basis for cardiac arrhythmias. Traditionally, most arrhythmias had been identified in terms of their appearance in the electrocardiogram (sinus tachycardia, a–v junctional tachycardia, ventricular tachycardia) and treatment was based largely on prior experience rather than on an understanding of basic mechanisms. Many of the possible mechanisms had been revealed at the level of the underlying phenomenology. Reentrant excitation or circus movement was well known and accepted as a cause of atrial flutter [12] as well as some ventricular tachycardias. Other arrhythmias were assumed to result from ectopic impulse generation. However, the electrophysiological basis for any of these events was a mystery. An effort to base arrhythmia identification on cellular electrophysiologic mechanism was made by Cranefield and Hoffman [13]. They classified arrhythmias under two major headings: abnormal impulse generation and abnormal impulse conduction. Impulse generation was either automatic or triggered. Automatic rhythms resulted either from the normal mechanism — phase 4 depolarization — or from an abnormal mechanism. Triggered rhythms were caused by either early or delayed afterdepolarizations. Impulse generation here depended on a prior impulse and thus was not automatic. Reentrant rhythms were subdivided in terms of ordered or random reentry. For the former the impulse traversed a defined path whereas for the latter there were multiple wavefronts traveling over varying paths, i.e. fibrillation. During reentry it was assumed that the impulse might be either a normal fast response or a slow response. Finally, it was suggested the some arrhythmias were caused by simultaneous abnormalities of impulse generation and conduction. This seemed likely in that phase 4 depolarization not only would lead to automatic firing but also would partially inactivate fast channels and thus reduce the safety factor for conduction. A major emphasis made by this classification was that identification of the basic mechanism for an arrhythmia should permit a more rational approach to therapy. This idea was not generally accepted until many years later [14]. This classification was the outgrowth of a large number of studies on isolated preparations of cardiac tissue and subsequently was somewhat modified and refined in terms of additional findings. The possibility of ectopic automatic rhythms was based on the demonstration of phase 4 depolarization throughout the His–Purkinje system and also the finding that phase 4 depolarization could result in automatic firing at multiple sites in the atria [9]. Afterdepolarizations were known from early records of monophasic potentials but the demonstration that both early and late afterdepolarizations were in fact the cause of selected arrhythmias was provided by microelectrode studies; this work is extensively reviewed by Cranefield and Aronson [15]. Delayed afterdepolarizations were shown to cause digitalis-induced junctional and ventricular arrhythmias [16,17] and early afterdepolarizations were the cause of quinidine-induced ventricular arrhythmias associated with delayed repolarization [18].

Traditionally it had been assumed that the response of cardiac tissue to stimulation was all-or-none in nature, This concept was corrected through recordings obtained from the junction between peripheral Purkinje fibers and ventricular muscle [19]. In the Purkinje fibers the action potential duration was considerably longer than in muscle. As a consequence premature impulses in the ventricular muscle encountered only partially repolarized Purkinje fibers and generated graded responses during the latter part of phase 3. These graded responses were either non-propagated or propagated slowly, often after considerable delay, and were prone to block. Graded responses also were recorded from partially depolarized fibers in the border zone of ischemic myocardium in intact hearts [20] and in a variety of conditions in which the resting transmembrane potential was reduced. This primary basis for slowed conduction of the cardiac impulse explained a number of conduction abnormalities and advanced understanding of reentry. The subsequent demonstration in isolated preparations of atrial tissue of leading circle reentry provided another major conceptual advance [21]. These studies showed that the area of block required for circus movement could be purely functional in nature and induced by the circulating impulse itself. Leading circle reentry differed from reentry around a permanently inexcitable barrier in terms of responses to premature impulses, overdrive and drugs. A rather special mechanism for reentrant excitation—reflection — was demonstrated by Cranefield [22] and also by Antzelevitch et al. [23]. In this case measurements of transmembrane potentials of Purkinje fibers with microelectrodes showed that when the impulse propagated up to a site of very slow conduction or partial block a new impulse was generated proximal to this site and propagated in the retrograde direction. The new impulse arose because the slowly propagating action potential generated depolarizing currents which, because of the delay, excited proximal fibers after they had sufficiently repolarized. One important conclusion from this demonstration was that reentrant excitation could occur within a very small volume of tissue.

That there was a vulnerable period for initiation of ventricular fibrillation by a single stimulus was well established [24]. Early in the 1950s Brooks' laboratory undertook a new study of the recovery of excitability of the canine heart using what then were modern methods [25]. Two findings were noteworthy. One was the demonstration of a dip in the curve depicting the recovery of excitability during repolarization. The other was a new finding for the vulnerable period. Typically, increasingly strong stimuli applied during the vulnerable period elicited first single responses, then multiple responses and then fibrillation. The new finding was that a further increase in stimulus strength caused no response. Both the dip in the excitability curve and the ‘no response’ phenomenon were shown to be associated with the anodal electrode [25]. This demonstration of anodal excitation and its failure at high intensities during phase 3 awaited measurements with the microelectrode for explanation. Weidmann [26] applied long-duration hyperpolarizing (anodal) current pulses during phases 2 and 3 and noted that sufficiently strong currents accelerated repolarization and caused break excitation. Still stronger stimuli caused complete repolarization and no break excitation. A threshold for all-or-nothing repolarization was thus demonstrated. These findings were extended by Cranefield and Hoffman and employed to explain not only the dip and the no response phenomena but also the responses of the ventricles to the strong shocks employed for defibrillation.

The first microelectrode study of the mechanism of action of antiarrhythmics was performed by Weidmann [27] and demonstrated the effects of the local anesthetic cocaine on voltage-dependent availability of the sodium channel as indicated by changes in the maximum rate of depolarization during phase 0 of the Purkinje fiber action potential. Cocaine decreased the maximum rate of depolarization of responses initiated at the normal resting potential and shifted the voltage dependence in a hyperpolarizing direction so that the upstroke of premature responses was greatly slowed and responses early during phase 3 became impossible. The same study also showed that an increase in extracellular calcium concentration had an opposite effect, shifting the relationship towards more depolarized potentials. As a result responses could be elicited from quite depolarized voltages during phase 3. Other investigators extended these observations on the effects of local anesthetics on sodium current to the major Class I drugs and also showed depressant effects on inward calcium current and delayed rectifier currents. One study on the procaine amide metabolite N-acetyl procaineamide [28] showed that it caused significant action potential prolongation without any effect on the action potential upstroke, This observation led, after some years, to the development of major additions to the class III antiarrhythmics that were more or less selective blockers of the delayed rectifier potassium currents.

Another major advance made possible by the use of the microelectrode was the development of methods to employ the voltage-clamp technique to study cardiac tissues. In this method one intracellular electrode is used to record the transmembrane potential and the second electrode is used to pass current across the membrane and thus control the transmembrane potential. The associated electronic circuitry allows the membrane potential to be clamped at any desired value and the relationship between membrane potential and ionic currents to be investigated. Cardiac tissues are not well-suited to two microelectrode voltage clamp experiments of this sort because of their syncytial nature and complex extracellular structure. Precise measurements awaited the development of methods to isolate single myocytes and study them with patch electrodes [29]. Nevertheless a great amount of crucially important information was obtained through experiments employing two microelectrode voltage clamp. Among the important discoveries were the description of the voltage- and time-dependent properties of sodium current activation and inactivation, the identification of the inward calcium currents as the generators of the slow response action potentials of the sinoatrial and atrio–ventricular nodes, the identification of the two components of the calcium current, L and T, and their role in maintenance of the action potential plateau, excitation–contraction coupling and sinus node pacemaker activity. Equally important was the demonstration of the two components of the delayed rectifier current and their role in repolarization and class III antiarrhythmic drug action. Finally, the identification of the two components of the transient outward current contributed to a better understanding of the basis for phase I of the action potential and also the importance of phase I to the level of the action potential plateau and the voltage–time course of repolarization. A related technique was the development of ion-sensitive microelectrodes. When paired with a standard microelectrode a potassium-sensitive electrode permitted measurement of activity-dependent changes in potassium levels in intercellular clefts [30] and the larger increases in extracellular potassium concentration caused by local ischemia [31]. Studies of this sort showed the extent to which the immediate extracellular environment of cardiac myocytes was controlled by transmembrane ionic fluxes. Use of the intracellular microelectrode to record from either isolated preparations or intact hearts provided much new information on the electrophysiological consequences of ischemia and infarction and to a better understanding of the genesis of early and late arrhythmias [32].

Microelectrodes also were used to record from preparations of normal and diseased human hearts and to a large extent conformed what had been learned through studies on tissues from laboratory animals. Microelectrodes, used either to record transmembrane potentials or to conduct voltage clamp experiments, provided much new information on the consequences of activation of cardiac autonomic receptors and explained the action of acetylcholine to slow the sinus node, shorten the atrial action potential and slow or block a–v nodal transmission: all were caused by an increase in potassium conductance. The microelectrode era waned with the development of methods to isolate single viable cardiac cells and study their electrical activity with patch electrodes. With this method it was possible to record the currents of single membrane channels, to exert quite precise voltage control of the membrane potential and to follow rapid transients. Also, through the use of isolated membrane patches either the inner or outer surface of the sarcolemma could be exposed to the experimental environment. Nevertheless, studies with microelectrodes continued in many laboratories and provided new information such as the existence of M cells in the depth of the ventricular myocardium, cells with prolonged action potential durations that seem to be of major importance in causing gradients of repolarization and the generation of early afterdepolarizations [33]. Microelectrodes most likely will continue to be used to study isolated preparations of cardiac muscle from normal and diseased hearts when information on the behavior of the syncytium, rather than the single myocyte, is required. In summary, the development of the microelectrode brought understanding of cardiac electrophysiology out of the dark ages and provided immensely important new knowledge of normal and abnormal cardiac electrical activity, the mechanisms for cardiac arrhythmias and the mode of action of many cardiac drugs.


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