Download Electrophysiological Study of Human Heart Muscle

Electrophysiological Study of Human
Heart Muscle
By Wolfgang Trautwein, M.D., Donald G. Kassebaum, M.D.,
Russell M. Nelson, M.D., Ph.D., and Hans H. Hecht, M.D.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
• Attempts to record membrane potentials
from the human heart exposed during surgery
have been reported by Woodbury et al.1 and
Bromberger-Barnea et al.2 In these studies,
rather large variation in the amplitude and
time course of the potentials was observed,
perhaps related to the circumstances of working with flexibly mounted micro-electrodes on
an exposed, contracting heart. The present
experiments were undertaken on the strength
of the evidence that records obtained from
suitably prepared and maintained tissue
preparations excised from other mammalian
hearts do not differ from records made in
situ.- 4 Membrane potential changes of small
preparations of human ventricular and atrial
tissue, excised during open-heart surgery,
were recorded in vitro, and the effects of
physical and chemical influences on the resting and action potentials of single myocardial
fibers were studied to test whether the basic
concepts of the mechanisms underlying the
action potential, automaticity and transmitter
effects, derived from investigation of other
cardiac tissue, are applicable to the human
heart.
Methods
Small pieces of pectinate muscle from the right
atrium, or trabeculae carneae from the right ventricle, were excised from the hearts of patients
undergoing1 corrective open-heart surgery. Atrial
specimens were obtained from five patients, aged
2, 10, 28, 32, and 53 years. The patients aged 2S
and 53 had aortic incompetence and aortic stenosis,
respectively; the others had atrial septal defects.
Ventricular tissue was obtained from two patients,
aged 4 and 6 years, who had ventricular septal
defects. Immediately after excision, the tissues
were immersed in cool Tyrode's solution (temperaProm the Department of Medicine, University of
Utah College of Medicine, Salt Lake City, Utah.
Supported by grants from the Utah Heart Association and U. S. Public Health Service (nos. HTS
5150 and HT 250).
Received for publication October 2, 1961.
306
ture about 30 C.) and carried from the operating
room to the laboratory. Small muscle strips were
then prepared by dissection and fixed in a tissue
chamber through which Tyrode's solution flowed at
a rate of 4 ml./min.; this was accomplished within
30 minutes after the tissue was excised from the
heart. During the subsequent hnlf-hour, the tissue
was allowed to recover while stimulated electrically
at a rate of 20/min. In the further course of the
experiments, those preparations which did not beat
spontaneously were driven electrically with a
Grass stimulator at rates of 30 and 60/min., for
ventricle and atrium, respectively.
The Tyrode's solution had the following composition (mM) : Na, 153; K, 2.7; Ca, 1.8; Mg, 1.05;
Cl, 145; HCOa, 13.5; H,P0 4 , 2.4; a.nd glucose,
5.5. The temperature of the bathing fluid was 36
to 38 C. The solution was equilibrated with a gas
mixture of 95 per cent Oo and 5 per cent COo,
providing a. pH between 7.2 and 7.4. A sodiumdeficient solution, containing only 16 mM NaCl,
was prepared by substituting an equivalent amount
of choline chloride for the excluded NaCl.
The acetylcholine chloride and epinephrine hydrochloride solutions used in these experiments
were delivered into the tissue bath with a syringe
and allowed to flow aJong the preparation in the
stream of Tyrode's solution, having contact with
the preparation for only a few seconds. The concentrations of acetylcholine and epinoplirine, so
diluted, were about 10"° and KH Gm./ml., respectively.
Membrane potentials were measured with glass
micropipettes filled with 3M KC1, having tip diameters smaller than 0.5 YU, and resistances between 8 and 20 megohms. The recording system
consisted of a cathode follower with negative
capacitance (time constant 10 to 50 /xsec.) and a
Tektronix twin-beam type 502 oscilloscope.
Results
VENTRICULAR TISSUE
Resting and Action Potentials
An action potential recorded from a ventricular muscle fiber can be seen in figure 1.
The rapid upstroke is followed by a plateau
at a positive potential leading into repolarization to the resting potential. From 24 successful impalements at multiple sites in two
preparations, resting potentials ranged beCirculation Research, Volume X, March 196S
307
HUMAN HEART MUSCLE
+ 20r
O
mV
40 mV
-1OOL
—
0-1 sec
FIGURE 1
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Action potential of a single ventricular fiber. The
preparation was driven electrically at a rate of
30/min. (note stimulus artifact). The zero line
was recorded during the same exposure when the
micro-electrode was withdrawn.
tween -71 and -95 mv. The mean ventricular
resting potential was -87 mv. (S.D. ± 5, S.B.
±1). The action potentials varied between 92
and 125 mv., with a mean of 115 mv. (S.D. ±
9, S.E. ± 2).
Upstroke and Conduction Velocity
The sigmoid upstroke of the action potential (fig. 2) rose from 5 to 95 per cent of its
amplitude in less than 1 msec. When the fiber
membrane was gradually depolarized by increasing the extracellular potassium concentration, the upstroke velocity was successively
reduced (cf. Weidmann5). Conduction velocity was determined five times in one preparation. Two electrodes were impaled in a fiber
within a longitudinal distance varying between 0.15 and 2.5 mm. The mean conduction
velocity, determined from the latency between the known distances (fig. 2), was 1.3
M./sec, with calculated values ranging between 1 and 1.6 M./sec. The variation was
not related to the distance between the recording electrodes. No action potentials with
prominent spikes, typical of the faster conducting Purkinje system, could be detected
in the areas where the conduction velocity
was determined (cf. Draper and Mya-Tu°).
This relatively high conduction velocity, compared to that measured in the dog ventricle,7
may be due, in part, to hypertrophy of the
fibers in the human preparations obtained
from hearts having ventricular septal defects.
Histological definition of the fiber diameter
Circulation Research, Volume X, March 1961
1 msec
FIGURE 2
Upstrokes of action potentials recorded simultaneously tvith two intracellular electrodes impaled at
a- distance of 2.5 mm. longitudinally in a ventricular muscle fiber. The slight distortion of the upstroke on the right is due to the longer time constant of the second recording system.
was not done, but ventricular hypertrophy
was apparent when the hearts were exposed
at surgery.
Effect of Reduced Extracellular Sodium
In the light of the sodium theory,8 it was
of interest to test the effect of reduced extracellular sodium concentration on the overshoot and on the rate of rise of the action
potential. No attempt was made to study this
quantitatively, but figure 3 (A) shows the effect of low external sodium in a qualitative
manner (cf. Draper and Weidmann,9 Brady
and Woodbury10). After the control recording, the normal Tyrode 's solution was replaced
by one containing only 16 mM sodium. The
overshoot and duration of the action potential
were reduced successively and the upstroke
velocity of the action potential was diminished (fig. 3B). The preparation became inexcitable after 15 minutes. The smallest
action potential in figure 3 (A) is one of the
last responses to a strong stimulus and is
preceded by a large stimulus artifact. As in
other mammalian cardiac tissue, the effect of
sodium depletion was quickly reversed when
308
TRAUTWEIN, KASSEBAUM, NELSON, HECHT
+ 3GV
-I00 1
O.I sec
FIGURE 3
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
(A) Superiinposed tracings of three action potentials recorded with an intracellular electrode from
a rentricular muscle fiber. The largest action potential was recorded when Tyrode's solution bathed
the preparation. The two smaller action potentials
were recorded 7 and 10 minutes after the Tyrode's
solution (153 mM sodium) was replaced by one
containing only 16 mM sodium. The stimulus intensity had to be greatly increased to reach threshold in low sodium (note the large stimulus artifact). (B) Tracings of upstrokes shoiving the
effect of reduction in extracellular sodium concentration. The left upstroke icas recorded in Tyrode's
solution. The upstroke on the right sho^vs a reduced rise velocity and was recorded in the loiu
sodium (16 mM) bathing solution.
Tyrode 's solution was readmitted to the bathing chamber.
Effects of Rate and Anoxia on the Plateau
Figure 4 depicts the shortening of the
duration of the action potential when the
stimulus rate was increased. The longest action potential was recorded at a rate of 24/
iniii., after which the rate of stimulation was
increased in five steps and the corresponding
action potentials superimposed on film. Figure 5 shows the effect of anoxia on the action
potential. When, the Tyrode's solution was
equilibrated with nitrogen, loss of plateau
and shortening of the action potential duration occurred within 15 minutes. The shortest
action potential, after 20 minutes nitrogenanoxia, rises from a slightly lower potential
level, causing a small reduction in overshoot.
ATRIAL TISSUE
111 contrast to ventricular muscle, it was
more difficult to obtain recovery of excised
atrial tissue. Two preparations showed oscillatory potential changes around low potential
levels of -30 to -40 mv. Another preparation
also exhibited spontaneous activity, and po-
-100L
0-1 sec
FIGURE 4
Superimposed, action potentials recorded during a
single impalement of a ventricular fiber, showing
the reduction in action potential duration as the
stimulus rate was increased in the following steps:
24, 48, 60, 96, 120, and 162/min. Little change
was recorded when the stimulus rate was increased
in the step from 24 to 48/min. An action potential
was recorded after adaptation to each change in
driving rate.
tential cycles with prominent diastolie depolarizations were recorded at all sites of electrode impalement (fig. 6). The maximum
diastolic potentials in this preparation varied
between -55 and -60 mv. These three preparations were excited from multiple foci and
showed asynchronous small movements, sometimes at a fast rate, characteristic of fibrillation. Such preparations were excitable only
early in diastole, when the membrane potential was relatively high (-50 to -55 mv.).
Responses early in diastole had a larger amplitude and were often conducted through the
entire muscle preparation, causing synchronized activity for one cycle. Responses to
electrical stimuli later in diastole were of
smaller amplitude and invaded only part of
the preparation.
Since oscillatory potential cycles of low
amplitude and low membrane potentials are
frequently observed in deteriorated or drugtreated preparations of other mammalian
hearts, it is unlikely that they represent the
normal physiological state.
Resting and Action Potentials
Only two preparations had reasonably high
resting and action potentials and constant
diastolic membrane potentials. These were
obtained from patients, aged 6 and 10 years,
Circulation Research, Volume X, March 196S
HUMAN HEART MUSCLE
+ 30r
0-1 sec
FIGURE
-100L
0-1 sec
7
(A) Action potential of a single atrial muscle
fiber. (B) Action potential of single atrial fiber
showing prominent spike and plateau with longer
action potential duration. The stimulus frequency
in both cases was 60/min.
FIGURE 5
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Three superimposed action potentials recorded
during one impalement in a ventricular fiber when
the Tyrode's solution was equilibrated with nitrogen. The stimulus rate was constant at 60/min.
The longest action potential was the control, and
the other two were recorded after 15 and 20
minutes of nitrogen-anoxia. The shortest action
potential starts from a slightly lower potential
level, causing a small reduction in overshoot.
0
mV
0
mV
-100 L_
0 1 sec
FIGURE 8
-60
1 sec
FIGURE 6
Action potentials of loiu amplitude recorded from
a spontaneously beating atrial muscle fiber. Prominent pacemaker potentials are seen.
with atrial septal defects. The resting potentials of 17 successful impalements at multiple
sites varied between -60 and -80 mv., and
the mean was -70 mv. (S.D. ± 6, S.E. ± 1).
Prom 19 successful impalements, the action
potentials ranged between 64 and 90 mv., and
the mean was 75 mv. (S.D. ± 8, S.E. ± 2).
Figure 7 (A) shows an action potential of
contour similar to that of atrial fibers of other
mammals, having no plateau and repolarization following right after the upstroke. Figure 7(B) depicts an action potential with
prominent spike and plateau at a negative
membrane potential. This latter type of action
potential was found commonly, and had a
longer duration, although the resting and
Circulation Research, Volume X, March 196B
Superimposed action potentials of an atrial fiber
recorded during the same electrode impalement before and after application of acetylcholine (10'"
Gm./ml.). Note the increase in resting potential
and, shortening of the action potential duration.
action potentials differed little from the recorded potential cycle of more normal contour.
Effect of Acetylcholine
Figure 8 shows the effect of acetylcholine
on an electrically driven preparation. The
resting potential increased to a level of
greater negativity, the amplitude of the spike
increased, and the action potential duration
shortened.
Effect of Acetylcholine and Epinephrine
on the Ectopic Pacemaker
On two occasions the preparations began
to beat spontaneously in the bath, and were
excited from a single pacemaker which could
be located by multiple impalements at different sites in the preparation. At the locus of
the pacemaker, typical pacemaker potentials
were recorded, as shown in figures 9 and 10.
310
TRAUTWEIN, KASSEBAUM, NELSON, HECHT
H
FIGURE 9
6
Eject of acetylcholine (10' Gm./ml.) on the membrane potential of an ectopio pacemaker in the
atrium. The application of acetylcholine is indicated by the arrow in (A). Further description
in text.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
When acetylcholine was applied (arrow in
fig. 9A), the ectopic pacemaker was strongly
inhibited. The arrival of the drug at the site
of electrode impalement caused a suppression
of the pacemaker potential, the membrane potential increased and the preparation was
arrested for about 40 seconds. After this, the
effect of acetylcholine gradually disappeared,
and slow depolai-ization occurred (fig. 9, B
an d C). The rate subsequently increased to
that of the control (fig. 9D).
When the rate of the ectopic pacemaker
slowed in the course of the experiment, the
effect of epinephrine on spontaneity was
tested (fig. 10). This also caused an initial
suppression of the pacemaker, followed by
arrest at the level of the antecedent maximum
diastolic membrane potential (fig. 10, A and
B). The inhibitory period lasted about 30
seconds; then the pacemaker resumed beating
with increasing rate. The first three action
potentials when the beat resumed are seen in
figure 10(C) and show a successive increase
in the amplitude of the spike. The pacemaker
subsequently reached a high rate, which was
maintained for several minutes. Figure 10(D)
shows the excitatory period of epinephrine,
during which the maximum diastolic potential
was higher and the overshoot consequently
greater than before epinephrine. An initial
inhibitory effect was observed with each application of epinephrine in both of the preparations in which an ectopic pacemaker
developed.
Discussion
The mean resting and action potentials of
human ventricular fibers in vitro are higher
FIGURE 10
Effect of epinephrine (10~5 Gm./ml.) on the membrane potential of an ectopic pacemaker in the
atrium. Epinephrine was applied 10 seconds before the first recording (A). Further explanation
in text.
than those recorded from, the human heart in
situ.2 It should be noted, however, that the
largest potentials recorded in situ are close
to those recorded in the present study. This
suggests that the condition of the tissue in
either case does not differ appreciably, insofar as the membrane properties are concerned.
The shape of the action potential recorded in
vitro, with long plateau, is corroborative evidence of the good condition of the preparation.
As with other mammalian cardiac fibers,
the upstroke velocity of the action potential
depends on the external sodium concentration,
and excitability is lost when the sodium driving force is critically reduced.0'10
The changes in action potential effected by
alterations in beat-frequency or by anoxia
are like those described for other mammalian
cardiac fibers11"13 and reveal the sensitivity
of the plateau duration to these factors.
The conduction velocity in human ventricle
was found to be around 30 per cent higher
than that determined by Hoffman in dog.7
The limited number of measurements must be
considered in any attempt to reconcile this
disparity. However, if the difference be real,
it may be related to muscular hypertrophy,
since the conduction velocity is proportional
to the square root of the fiber radius (cf.
Katz14).
In contrast to ventricular muscle strips, it
was more difficult to obtain recovery and survival of excised atrial tissue. While an explanation for this difference in viability is
beyond the scope of the present report, several considerations seem pertinent. The two
muscle specimens obtained from the hearts
Circulation Research, Volumo X, March 1968
311
HUMAN HEART MUSCLE
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
of young patients (aged 6 to 10 years) gave
satisfactory results, in contrast to the other
three preparations excised from the hearts of
adults having long-standing heart disease,
two of whom were receiving digitalis for cardiac failure. The fibers of these latter preparations were difficult to impale and contained
abundant connective tissue. Of special interest is the related observation that the fibers
of atrial tissue excised from dog show a tendency to depolarize when they are not stimulated in vitro, an effect which is clearly reversible if the preparation is stimulated while
it is still excitable.15
The human atrial resting and action potentials recorded in this study are lower than
those found in dog10 and rabbit.17 While action potentials with plateaus may be observed
in dog atria, the plateaus characterizing the
second type of action potential in the present
study seem unusually prominent, and suggest
a basic disturbance in the mechanism underlying repolarization. It is probable that the
membrane conductance for potassium was reduced in these atrial fibers, especially in view
of the striking hyperpolarization caused by
acetylcholine. It is possible that these fibers
lost potassium, whereby the driving force for
repolarization was reduced. An alternate
mechanism responsible for the prolonged
plateau would entail an increased sodium
conductance of the membrane. A large sodium
conductance in such fibers could underlie
their tendency to spontaneous rhythmicity.18
The ectopic pacemaker activity in the atrial
preparations was strongly inhibited by acetylcholine. The mechanism of inhibition, by
virtue of increased potassium conductance of
the membrane, is undoubtedly like that in
other mammalian hearts.35
The initial, transient hyperpolarization and
inhibition produced by epinephrine was surprising. It is of related interest that hyperpolarization can also be seen in the arrested
dog atrium in the presence of epinephrine,10
an effect uninfluenced by atropine. In the
excised sino-atrial node of dog or rabbit,
epinephrine never causes inhibition, but
shows only an excitatory effect.
Circidation Research, Volume X, March 1968
Summary
Membrane potential changes in human
ventricular and atrial muscle, excised from
patients undergoing open-heart surgery, were
recorded by micro-electrodes in vitro.
Mean ventricular resting and action potentials were —87 mv. and 115 mv., respectively.
The mean atrial resting potential was —70
mv., mean action potential 75 mv. Two forms
of atrial action potential were found, one
having conventional contour, the other with
prominent spike and plateau. A disturbance
in repolarization is believed to underlie the
latter type of atrial potential cycle.
The relation between the upstroke velocity
of the action potential and the extracellular
sodium concentration and membrane potential was shown to be similar to that in other
mammalian cardiac tissue. The mean conduction velocity determined in ventricular fibers
(1.3 M./see.) was somewhat greater than that
of the dog, and the possible relationship to
hypertrophy of the cardiac fibers in the
preparations studied is described.
The effect of increased rate and anoxia in
reducing the action potential duration is like
that found in the hearts of other mammals.
The conductance type of inhibition was
produced by acetylcholine in spontaneously
beating atrial tissue. The excitatory effect of
epinephrine was preceded by a transitory
inhibition.
The basic mechanisms underlying the
action potential, automaticity and transmitter effects, derived from investigation of
other mammalian cardiac tissue are applicable to the human heart.
References
1. WOODBURY, J. W., LEE, J., BRADY, A. J., AND
MERENDINO, K. A.: Transmembrane potentials
from the human heart. Circulation Kesearch
6: 179, 1957.
2. BROMBERGER-BARNEA, B., CALDINI, P., AND
WITTENSTEIN, G. J.: Transmembrane potentials
of the normal and hypothermie human heart.
Circulation Research 7: 138, 1959.
3. TRAUTWEIN, W., UND ZINK, K.: tJber Membran-
iind Aktionspotential, einzelner Myokardfasern des Kalt- und WarmbHiterherzens. Arch,
ges. Physiol. 256: 68, 1952.
312
TRAUTWEIN, KASSEBAUM, NELSON, HECHT
4. HOFFMAN, B. F., AND SUCKLING, E. B.: Cellular
potentials of intact mammalian hearts. Am.
J. Physiol. 170: 357, 1952.
5. WEIDMANN, S.: Effect of the cardiac membrane
potential on the rapid availability of the
sodium-carrying system. J. Physiol. 127: 213,
1955.
6. DRAPER, M. H., AND MYA-TU, M.: Comparison
of the conduction velocity in cardiac tissues
of various mammals. J. Exper. Physiol. 64:
92, 1959.
7. HOFrMAN,
B.
F.,
AND CBANEMELD, P.
F.:
Electrophysiology of the Heart. New York,
McGraw-Hill Book Co., 1960.
3. HODGKIN, A. L., AND HUXLEY, A. F . : Quantita-
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
tive description of membrane current and its
application to conduction and excitation in
nerve. J. Physiol. 117: 500, 1952.
9. DRAPER, M. H., AND WEIDMANN, S.:
Cardiac
resting and action potentials recorded with an
intracellular electrode. J. Physiol. 115: 74,
1951.
12.
TRAUTWEIN, W., UND DUDEL, J.: Aktionspotential
und Mechanogramm des Warmbliiterherzmuskels als Funktion der Schlagfrequenz. Arch,
ges. Physiol. 260: 23, 1954.
13. TRAUTWEIN, W., GOTTSTEIN, U., UND DUDEL, J.:
Der Aktionsstrom der Myokardfaser im Sauerstoffmangel. Arch. ges. Physiol. 260: 40, 1954.
14. KATZ, B.: Electrical properties of the muscle
fiber membrane. Proc. Roy. Soc, London s.B
135: 506, 1948.
15. TRAUTWEIN, W.,
UND DUDEL, J.:
Hemmende
and "erregende" Wirkungen des Acotylcholins
am Warmbliiterherzen. Zur Frage der spontanen Erregungsbildung. Arch. ges. Physiol.
266: 653, 1958.
16. HOFFMAN, B. F., AND SUCKLING, E. E.: Cardiac
cellular potentials: Effect of vagal stimulation
and acetylcholine. Am. J. Physiol. 173: 312,
1953.
17. WEST, T. C.: Ultramicroelectrode recording from
the cardiac pacemaker. J. Pharmacol. & Exper.
Therap. 115: 283, 1955.
10. BRADY, A. J., AND WOODBURY, J. W.: Sodium-
18. TRAUTWEIN, W., AND KASSEBAUM, D. G.: On the
potassium hypothesis as the basis of electrical
activity in frog ventricle. J. Physiol. 154:
383, 1960.
mechanism of spontaneous impulse generation
in the pacemaker of the heart. J. Gen. Physiol.
45: 317, 196.1.
11. HOFFMAN, B. F., AND SUCKLING, E. E.:
Effect
of heart rate on cardiac membrane potentials
and the unipolar electrogram. Am. J. Physiol.
179: 123, 1954.
19. TRAUTWEIN, W.,
UND SCHMIDT, B.
F.:
Zur
Meinbranwirkung des Adrenalins an der
Hermuskelfaser. Arch. ges. Physiol. 271: 715,
1960.
Circulation Research, Volume X, March 196S
Electrophysiological Study of Human Heart Muscle
WOLFGANG TRAUTWEIN, DONALD G. KASSEBAUM, RUSSELL M. NELSON and HANS
H. HECHT
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Circ Res. 1962;10:306-312
doi: 10.1161/01.RES.10.3.306
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1962 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/10/3/306
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/