Effects of Extracellular Calcium and Sodium on

Depolarization-Induced Automaticity in Guinea Pig

Papillary Muscle
By Bertram G. Katzung

ABSTRACT
Regenerative discharge of action potentials is induced in mammalian papillary muscles
by passage of small depolarizing currents. In this paper, the effects of various extracellular
calcium and sodium concentrations and of tetrodotoxin on this phenomenon were studied
in guinea pig papillary muscles in a sucrose gap chamber. Phase 4 diastolic depolarization
was found to be associated with an increase in membrane resistance. The slope of phase
4 depolarization was decreased by reductions in extracellular calcium or sodium concen-
tration. The range of maximum diastolic potentials and the thresholds from which regener-
ative potentials arose were reduced, especially at the positive limit of potentials, by a re-
duction in either ion. It was concluded that both calcium and sodium influence diastolic
depolarization and participate in the regenerative action potentials of depolarization-
induced ventricular automaticity.

• The occurrence of automaticity in mammalian
ventricular myocardial fibers when they are sub-
jected to small depolarizing currents has recently
been reported from this laboratory (1, 2). This
depolarization-induced automaticity appears to re-
semble the phenomenon previously reported in
mammalian Purkinje fibers (3-5), the chick em-
bryo heart (6), and the frog atrium (7, 8).
It is generally accepted that, in Purkinje fibers
Downloaded from http://ahajournals.org by on July 29, 2020
the recent work of Aronson and Cranefield (16) has
shown that Purkinje fibers soaked in Na-free solu-
tions for several hours are capable of phase 4 de-
polarization leading to Ca-dependent action poten-
tials.
In the case of automaticity induced by applica-
tion of current from an external source, two possi-
ble sources of depolarizing current can be postu-
lated: (1) an electrophoretic inflow of K ions from

and perhaps in all cardiac pacemakers, phase 4
depolarization occurs as the result of a decrease in
potassium (K) conductance in the presence of a
small depolarizing current (3, 9, 10). The nature of
the depolarizing current has not been completely
defined. Brooks and Lu (11) and West (12) in
recent reviews of sinoatrial nodal pacemaker physi-
ology have ascribed a role to calcium (Ca) ions.
In cultured chick heart cells, however, an elevation
of the extracellular calcium concentration, [Ca]o,
has no detectable effects (13). In spontaneously
firing Purkinje fibers, a reduction of the extracellu-
lar sodium concentration, [Na]o, reduces or abol-
ishes pacemaker activity, but changes in [Ca]o have
little effect on this variable (14, 15). Studies of de-
polarization-induced automaticity in Purkinje fi-
bers have led to suggestions that Na is primarily
responsible (3) or that both Na and Ca are involved
in phase 4 depolarizing currents (5). Furthermore,
From the Department of Pharmacology, University of Califor-
nia at San Francisco, San Francisco, California 94143.
This study was supported in part by U. S. Public Health
Service Grant HL 17452 from the National Heart and Lung In-
stitute.
Received October 31, 1975. Accepted for publication April 29,
1975.
the immediate current source, and (2) atransmem-
brane Na or Ca current. Preliminary experiments
(1, 2) have suggested that both Na and Ca are
involved in depolarization-induced automaticity in
guinea pig ventricular myocardial cells. In those
experiments, tetrodotoxin was used to diminish Na
current and verapamil was used as a Ca blocker. To
more clearly define the roles of Na and Ca in
ventricular automaticity, further experiments were
carried out in which the extracellular concentra-
tions of these ions were varied. Tetrodotoxin was
used as an inhibitor of the Na system (17, 18).
Methods
Guinea pigs (250-350g) were killed by cervical disloca-
tion. Papillary muscles (5 mm long x 0.6-1.0 mm in
diameter) were rapidly excised from the hearts and
mounted in a sucrose gap chamber of the type described
by Reuter and Scholtz (19). The tip of the papillary
muscle in the test chamber was limited to 0.6-1.0 mm in
length. Impalements were obtained with standard glass
microelectrodes, and membrane potential was taken as
the potential difference between the intracellular elec-
trode and a similar extracellular microelectrode located
at the surface of the muscle. Constant-current pulses
were generated by the feedback arrangement shown in
Figure 1. Command pulses were derived from Grass
stimulators. This type of feedback clamping was superior

118 Circulation Research, Vol. 37, July 1975

 Ca A N D Na O N V E N T R I C U L A R A U T O M A T I C I T Y
COMMAND
J-L
INV.
FIGURE 1
Schematic diagram of the experimental apparatus. The muscle
chamber is shown in heavy black outline, divided into three
compartments (T = Tyrode's solution perfusion and S = sucrose
perfusion) by two rubber membranes. The papillary muscle is
connected to the lever of a Grass FT.03 force transducer
(FORCE) at its tendinous end. Transmembrane potential is
monitored in the test compartment (bottom section of chamber)
by matched, differential capacity-neutralized followers
(POTENTIAL) and displayed on the oscilloscope at the left.
Current is injected into the top section of the chamber and
exits via the bottom, test compartment. Current thus passed
through the preparation is monitored by an operational am-
plifier (CURRENT), displayed, and fed back to the current
source (CLAMP) for comparison with the command pulses.
INV. = inverting operational amplifier.
to the common technique of using a large series resistor
Downloaded from http://ahajournals.org by on July 29, 2020
at the output of a stimulator without feedback, since it
provided truly rectangular current clamps under all
conditions. Current and transmembrane voltage were
displayed on an oscilloscope and recorded on film in
every experiment. In most experiments, the time differ-
ential of transmembrane potential (dV/dt), obtained by
standard operational amplifier techniques, was also
displayed and photographed. In a few experiments,
contractility was also measured using a Grass FT .03
isometric tension transducer. Observation of the prepa-
ration showed that contractile activity was usually
limited to the test chamber (bottom chamber section in
Fig. 1). The current injection chamber (top chamber
section in Fig. 1) was perfused throughout all experi-
ments with conventional bicarbonate Tyrode's solution
(NaCl 134 mM, KC1 5.0 mM, MgCl2 1.05 mM, CaCl2 1.8
mM, NaHCO 3 11.0 mM, NaH 2 PC\ 0.42 mM, and glucose
5.5 mM) which was bubbled with 5% CO2-95% O 2 . The
middle (sucrose) chamber section was perfused with
isotonic sucrose solution (300 mM) containing 5.5 mM
glucose and bubbled with 100% O2. The test compart-
ment was perfused with either bicarbonate Tyrode's
solution as described or Tris-buffered Tyrode's solution
in which NaHCO 3 and NaH 2 P0 4 were replaced with 5.0
mM Tris-hydroxymethylamine methane. Tris solutions
were titrated with HC1 to pH 7.2 (similar to the pH of the
bicarbonate Tyrode's solution at 36°C) and bubbled
with 100% O2. Solutions buffered at pH 7.4 gave identical
results. Variations in the Na concentration of the perfu-
sate were obtained by replacing various fractions of the
NaCl with isosmotic quantities of choline chloride.
Atropine sulfate was added to choline-containing solu-
Circulation Research, Vol. 37, July 1975
119
tions in the proportion of 1 mg atropine sulfate/140 mM
choline. A few experiments were also carried out in which
sucrose rather than choline chloride isosmotically re-
placed NaCl. No difference between the choline and the
sucrose results could be detected. [Ca]o was varied by
changing the amount of CaCl2 added without compensa-
tion for the relatively small variations in osmotic pres-
sure which resulted. All Ca variation experiments were
carried out using Tris-buffered solution. Tetrodotoxin-
containing solutions were made by dissolving 1 mgof the
lyophilized material (Calbiochem) in 50 or 100 ml of
Tris-buffered Tyrode's solution. Temperature was moni-
tored with a thermistor probe located in the anterior
chamber close to the preparation and was maintained at
36°C.
Experiments were carried out by stimulating the
muscle initially with short (2-5 msec) pulses 1-1.5 times
threshold at 0.5 Hz to evaluate action potential configu-
ration and ascertain that the normal resting potential
was completely stable. Then, current clamps 1-2 seconds
in duration were applied at a rate of 0.05 Hz starting at
subthreshold intensity and incrementing the magnitude
of the pulses. Test solutions were then perfused through
the anterior chamber, and the procedure was repeated
until a new steady state was defined. Generally, achieve-
ment of a new steady state required 20-40 minutes for
changes in [Ca]o and 15-30 minutes for changes in [Na]o.
Effects of tetrodotoxin were usually stable after 5-10
minutes but were checked for at least 20 minutes. A
washout in normal Tyrode's solution was then at-
tempted; if it was successful, further test solutions were
studied. In all cases, continuous single-cell impalements
were used to compare control and test parameters.
Results
Figure 2 illustrates some of the features of de-
polarization-induced automaticity in guinea pig
ventricular myocardial cells. Conventional short
stimuli elicited typical action potentials with flat
phase 4 intervals (section 1). Long current pulses of
increasing intensity led to progressively greater de-
polarization resulting in, first, a single elicited ac-
tion potential followed by a relatively stable but
depolarized potential (section 2) and then phase 4
depolarization leading to repetitive "spontaneous"
action potentials (section 3). There was a gradual
positive shift in threshold and a decrease in phase 0
dV/dt as depolarization was increased. As shown in
sections 3 and 4, phase 4 depolarization developing
after the most negative maximum diastolic poten-
tials was slow and frequently quite linear. How-
ever, with even small shifts to less negative max-
imum diastolic potentials (higher current intensi-
ties), phase 4 became progressively greater and
curvilinear (sections 5-8). At maximum diastolic
potentials more positive than —30 to —45 mv, a
slowing of the repetition rate was usually observed
(section 8). At maximum diastolic potentials posi-
tive to -10 mv, responses were usually limited to
 120 KATZUNG

20/JA
Or
•V
-50L
\ — V/S

1 \ 6 20juA

mV
-50
0
r LL
I I
T-
I V/S

20/JA

V/S

FIGURE 2
Typical series of records from a guinea pig papillary muscle in bicarbonate Tyrode's solution. Top
trace = current, second trace = transmembrane potential, third trace = dV/dt, fourth trace = time,
Downloaded from http://ahajournals.org by on July 29, 2020

and bottom trace = contractile force. Calibration for dV/dt (V/S): 200 u/sec for sections 1-6 and 20
v/sec for sections 7-9. Time calibration: major intervals (all panels) = 100 msec and minor intervals
(section 1 only) = 20 msec. The current pulse in section 1 was 5 msec in duration; in sections 2-9, it
was 1400 msec in duration.

small damped oscillations (section 9). The relation-
ship of phase 4 depolarization slope to maximum
diastolic potential varied somewhat for different
muscles, but the biphasic relationship was similar
in all. Following release of the current clamp, a
current-dependent residual depolarization lasting
1-5 seconds was always seen.
To evaluate overall changes in membrane con-
ductance during depolarization-induced automa-
ticity, resistance was measured by injection of l-/ua
rectangular current pulses at 5 Hz. The voltage
deflections produced by the current pulses were
taken as being proportional to membrane resist-
ance. Membrane resistance measured at reduced
membrane potentials during depolarizing pulses
was considerably greater than resistance measured
before or after the current pulse, indicating the
presence of significant anomalous rectification.
When depolarization was sufficient to lead to
progressive phase 4 depolarization, a further pro-
gressive increase in membrane resistance was de-
tected during the pacemaker potential.
EFFECTS OF EXTRACELLULAR CALCIUM
The effects of changing [Ca]o were studied in 11
preparations. Typical results are illustrated in
Figure 3. Altering [Ca]o frequently changed the
maximum diastolic potential-applied current rela-
tionship. Since the automaticity resulting from the
applied current is more consistently related to the
maximum diastolic potential than it is to the
current, the sections shown in Figure 3 were
selected for comparison on the basis of equivalent
maximum diastolic potential alone. In every exper-
iment, the most obvious effects of decreasing [Ca]o
were a small decrease in the overshoot and dV/dt of
the initial elcited action potential and a marked
decrease in both variables in subsequent regenera-
tive action potentials at all diastolic potential
levels (Fig. 3, sections 5 and 6). Secondly, the range
of diastolic potentials from which action potentials
arose was constricted, especially at the positive
range limit (compare sections 3 and 6; Table 1). In
most preparations, slowing of repolarization (phase
3) was observed for action potentials occurring
Circulation Research, Vol. 37, July 1975
 Ca AND Na ON VENTRICULAR AUTOMATICITY 121

- |20>JA
CONTROL 0 A_
[Co] mV
-50
l-8mM
i — |v/s

[00] •\ -YT
0-45 mM
— |v/s

8
|20>uA
l*~J Ol
mV
5-4 mM _J V
|v/s

FIGURE 3
Effects of [Ca]o. 1-3: Control responses showing a conventional action potential and automaticity at
high and low maximum diastolic potentials. 4-6: Responses in low [Ca]o. 7-9: Effects of high [Ca]o-
Sections showing automaticity were chosen on the basis of maximum diastolic potentials equal to
those of the control sections (see text). Traces and time calibrations are the same as they are in Figure
2; contractile force is not shown. dV/dt calibration (V/S): 20 v/sec in sections 3, 6, and 9 and 200
v/sec in all other sections.

during long current pulses in low [Ca]o. There was increased overshoot and upstroke dV/dt for regen-
no significant shift in threshold relative to maxi- erative action potentials, a somewhat broader
Downloaded from http://ahajournals.org by on July 29, 2020

mum diastolic potential for the regenerative action
potentials (note the parallel changes in threshold
and maximum diastolic potential limits, Table 1).
Maximum phase 4 slope (i.e., between -40 and
-55 mv) was usually moderately decreased by
reduction of Ca concentration. At less negative
maximum diastolic potentials, it was consistently
decreased and regenerative action potentials were
frequently abolished (Fig. 3, section 6).
As shown in the last three sections of Figure 3,
increasing [Ca]0 from 1.8 to 5.4 mM caused an
range of maximum diastolic potentials from which
regenerative action potentials arose, and shorter
action potential durations. Elevation of [Ca]o re-
sulted in a slight increase in phase 4 slope in
approximately half of the muscles studied and a
decrease in the remainder (Fig. 3, sections 7 and 8).
EFFECTS OF EXTRACELLULAR SODIUM
The effects of reducing [Na]o were studied i.i six
preparations. Because prominent effects were pres-
ent in some muscles at 72 mM [Na]0 (50% of

TABLE 1

Effects of Reductions in Extracellular Calcium and Sodium on Regenerative Activity

Range of maximum diastolic potentials* Range of thresholdsf

Negative limit Positive limit For negative limit For positive limit
Condition (mv) (mv) (mv) (mv)
Control (1.8 mM [Ca]o) -75.4 -29.0 -67.3 -10.3
0.18 mM [Ca]o -67.3 -49.3 -60.3 -32.0
Net change +8.1 ±2.75(4) -20.3 ±2.75(4) +7.0 ± 2.2 (4) -21.7 ±9.4(4)
Control (145 mM [Na]o) -70.3 -24.8 -64.3 -9.5
72.5 mM [Na]o -62.5 -34.8 -46.5 -16.5
Net change +7.8 ± 1.0 (4) -10.0 ±3.4 (4) + 17.8 ±3.8 (4) -7.0 ±4.5 (4)

Values for net changes are means ± SE; number of muscles tested is given in parentheses.
* Most negative and most positive values of maximum diastolic potential from which regenerative action potentials arose,
t Thresholds of the regenerative action potentials arising from the most negative and most positive maximum diastolic potentials.
Circulation Research, Vol. 37, July 1975
 122
CONTROL \
mV
[No]
145 mM -50
[No] mV
72mM -50
[NaJ
36 mM
mV
-50
A =1
...A.
FIGURE 4
Effects of reduction of [Na]0. 1-3: Control. 4-6: 50% Na. 7-9: 25% Na. Traces and time calibra-
tions are the same as they are in Figure 2. dV/dt calibration (V/S): 200 u/sec in sections 1, 2, 4, 5,
and 7 and 20 v/sec in sections 3, 6, 8, and 9.
Downloaded from http://ahajournals.org by on July 29, 2020
normal) and in all at 36 mM and because rapidly
increasing contractile force made it difficult to
maintain impalements, no attempt was made to
reduce [Na]o below 25% of normal. Figure 4 illus-
trates the effect of a reduction in [Na]o in two
steps. Note that at 50% [Na]o, the major electrical
effects resembled those of Ca depletion, i.e., reduc-
tion of action potential overshoot and upstroke
dV/dt (sections 2 and 3 vs. 5 and 6). With reduction
of [Na]o to 25%, there was a marked positive shift
in the threshold potential associated with more
negative maximum diastolic potentials (section 8).
This change is also shown clearly in Table 1, in
which a moderately positive shift in the negative
limit of the maximum diastolic potential range
(+7.8 mv) was accompanied by a much larger shift
in the threshold associated with these action poten-
tials ( + 17.8 mv). As a result, an increase in the
interval between the first elicited action potential
and the second regenerative action potential was
consistently observed. A further loss of action
potential overshoot and upstroke dV/dt and a
marked increase in contractile force were also
observed. However, some phase 4 depolarization
still occurred.
The effects of [Ca]o and [Na]o reduction on
KATZUNG
20/iA
V/S
20JUA
-i. V/S
8
20»A
\ -
V/S
ZLZ.
phase 4 depolarization rate in a different prepara-
tion are shown graphically in Figure 5. At 50% re-
duction of either ion, peak depolarization rates
were moderately reduced, but there was little effect
at more negative membrane potentials. With fur-
ther reduction of Na to 25% of normal, regenerative
action potentials were abolished in this muscle and
only phase 4 depolarization was observed. How-
ever, even with reduction of [Ca]o to 10%, well-de-
fined regenerative action potentials persisted at in-
termediate membrane potentials, and phase 4 de-
polarization rates were fairly well maintained. At
less negative potentials however (positive to -45
mv), regenerative action potentials were abolished.
The effects of variations in [Ca]o and [Na]o on
action potential overshoot are graphed in Figure 6.
It appears that action potential overshoot is par-
tially dependent on the extracellular concentra-
tions of Na and Ca at all membrane potentials
sampled. The apparent dependence on [Na]o is
particularly interesting, since Imanishi (5) con-
cluded that for depolarization-induced automatic-
ity in Purkinje fibers Na did not play a significant
role at less negative maximum diastolic potentials.
However, he did not report overshoot data for his
low-Na experiments. There are several reasons for
Circulation Research, Vol. 37, July 1975
 Ca AND Na ON VENTRICULAR AUTOMATICITY 123

B.

-75 -65 -55 -45 -35 -25 -75 -65 -55 -45 -35 -25

Maximum Diastolic Potential (mV)

FIGURE 5

Effects of reduction of [Ca\, (A) and [Na]o (B) on the average slope of diastolic depolarization,
measured as shown in the inset in a single papillary muscle. Curves were drawn by eye. Solid circles,
both A and B = control Tris-Tyrode's solution ([Ca]o = 1.8 mM, [Na\, = 145 mM). Open triangles, B
= linear phase 4 slopes in 25% Na (36 mM); curvilinear phase 4 and regenerative spikes were abolished
Downloaded from http://ahajournals.org by on July 29, 2020

at this [Na]o in this preparation. ER = resting potential preceding a current clamp, MDP =
maximum diastolic potential, and THR = threshold potential.

caution in the interpretation of these plots. First,
action potential amplitudes are increased during
constant-current pulses by an amount equal to the
current-resistance product (I x R) across the
structures lying between the intra- and extracellu-
lar microelectrodes. This I x R increment probably
differs at different [Ca]o or [Na]o (unpublished
results). Second, application of the Nernst relation-
ship assumes that permeability to other charge-
carrying ions is negligible (20). As shown by Reuter
(21), even a low level of concurrent K permeability
results in deviation from the theoretical slope for
curves relating overshoot to extracellular concen-
trations.
EFFECTS OF TETRODOTOXIN
Consideration of the effects of extracellular Na
reduction leads to the conclusion that the results
shown in Figures 5 and 6 cannot all be attributed to
simple extracellular Na depletion. As suggested by
the dramatic increase in contractile force registered
in the experiment of Figure 4, [Na]o reduction is
associated with an increase in intracellular Ca (22,
23) which, in turn, will diminish the driving force
Circulation Research, Vol. 37, July 1975
for inward Ca current. Therefore, four preparations
were studied in which tetrodotoxin was used to
block Na currents. As indicated in Figure 7, this
agent resembled [Na]o reduction in that it greatly
reduced action potential Overshoot and upstroke
dV/dt (sections 4 and 5 vs. 1 and 2) and moved the
threshold to much more positive levels (section 5
vs. 2). However, tetrodotoxin had little effect on
the small regenerative action potentials occurring
at low membrane potentials (section 6 vs. 3).
There was little effect on contractile force (not
shown). When Ca in the perfusate was reduced to
0.18 mM while tetrodotoxin administration was
continued, phase 4 depolarization was almost elim-
inated, and all regenerative action potentials were
abolished (sections 8 and 9). However, the muscle
was still capable of active responses (section 10) if
it was depolarized to threshold by a short strong
stimulus (arrow) superimposed on the long current
pulse.
Discussion
The influence of resting potential on automatic-
ity in cells recognized as latent or overt pacemak-
 124 KATZUNG

50

40

,19*
30

20

10

• MDP - 6 0 to-65mV
O M D P - 5 0 t o - 5 5 mV
-10
x MDP - 4 2 to -48 mV
A MDP - 3 5 to - 4 0 mV

0I8 [Co " I rnM I-8 72-5

mM I45

FIGURE 6

Effects of [Ca]o and [Na]o on the overshooi amplitude of regenerative action potentials. Left: Mean
values from four preparations for which complete data were obtained in control (1.8 mM (Ca]o) and
10% (0.18 mM [Ca]o) Ca solutions. Vertical bars indicate +1 SE. S values indicate the slope of the
overshoot vs. [Ca]o in mv/tenfold increase in concentration (semilogarithmic plot). Broken line (S =
Downloaded from http://ahajournals.org by on July 29, 2020

30.6) = theoretical slope of a Ca electrode at 37°C. Right: Mean values from four preparations for
which complete data were obtained in control (145 mM [Na\,) and 50% (72.5 mM [NaD Na solutions.
Vertical bars indicate +1 SE. S values indicate the slope of the overshoot vs. [Na\> in mv/tenfold in-
crease in concentration (semilogarithmic plot). Broken line (S = 61.2) = theoretical slope of an Na
electrode at 37" C.

ers, e.g., Purkinje fibers (3-5, 24) and cultured
chick heart cells (6), has been well demonstrated.
Recently, evidence has accumulated which shows
PHASE 4 DEPOLARIZATION
that a number of normally quiescent cell types may
also undergo repetitive oscillations, often leading to
regenerative action potentials, when the resting
potential is reduced. This phenomenon has been
convincingly shown in frog atrial tissue by several
groups (7, 8). Using mammalian ventricular mus-
cle, Antoni and Tagtmeyer (25) have reported that
strong hyperpolarizing current pulses are followed
by a variable period of regenerative spike activity.
The present study, along with preceding prelimi-
nary reports (1, 2) shows that such activity can be
more readily induced in ventricular myocardium
by depolarizing currents.
Regenerative spike activity requires (1) a slow
depolarizing process which prevents the cell from
maintaining the maximum diastolic potential, (2) a
more rapid depolarizing (spike) process, and (3) a
repolarizing process which returns the cell to its
maximum diastolic potential (9, 26). The present
results yield some insight into the first two of these
processes.
It was first shown by Weidmann (27) that in
Purkinje fibers phase 4 depolarization is accom-
panied by increasing membrane resistance (calcu-
lated from the change in membrane potential
produced by small low-frequency current pulses).
The present results, utilizing a similar method,
indicate that a similar change in resistance accom-
panies diastolic depolarization in the ventricle.
However, lacking full voltage clamp analysis, it
cannot be stated whether this apparent conduct-
ance change is the cause or the result of the
observed phase 4 depolarization.
Considerable evidence from voltage clamp stud-
ies in Purkinje fibers indicates that deactivation of
one of two K conductances, gK2 or gX,, depend-
ing on the membrane potential, is responsible for
the phase 4 increase in membrane resistance and
phase 4 depolarization (28, 29). Several laboratories
Circulation Research, Vol. 37, July 1975
 Ca AND Na ON VENTRICULAR AUTOMATICITY 125

CONTROL
1
2
20juA
\ \ \ \ \ - —
3

0
UM,
mV
-50
A V/S

TTX 4 5
6 20/JA

\
A
0
mV
-50
V/S
i

TTX , Low Ca - 7 8 9 T 10
20^iA

A
0
mV ^—
-50 ' i
'j
V/S

FIGURE 7

Effects of tetrodotoxin (TTX) and calcium reduction. 1-3: Control Tris-Tyrode's solution. 4-6: 10~b
g/ml of tetrodotoxin in normal Tris-Tyrode's solution. 7-10: IO~* glml of tetrodotoxin in low-Ca
([Ca]o = 0.18 mu)Tris-Tyrode's solution. Traces and time calibrations are the same as they are in
Figure 2. dV/dt calibration (V/S): 200 v/sec in sections 1,2,4, and 5 and 20 vkec in all other sections.
Section 10 shows the effect of a 20-msec, 10-na pulse superimposed (arrow) on the long current pulse.

have defined an outward current in frog atrial mus- permeability or kinetics, as the sole cause of this
cle which closely resembles the IXl of Purkinje effect. For instance, reduction of [Ca]o might
fibers and is presumably involved in the depolariza- increase outward K current as it does in squid
Downloaded from http://ahajournals.org by on July 29, 2020

tion-induced automaticity demonstrable in this tis-
sue (7, 8, 30). In ventricular myocardium, time-
dependent outward currents vary for different
species (31) but have been demonstrated in guinea
pigs (32) and monkeys (33). It is not clear whether
the time dependence of these currents is the result
of a time-related conductance change or a change
in K gradient resulting from accumulation of K
ions in a diffusion-limited space (31). In relation to
the present results, the former mechanism is more
consistent with the observed increase in membrane
resistance during phase 4, but the latter mecha-
nism is supported by the residual depolarization
consistently observed following release of current
clamps. The absence of phase 4 depolarization in
the ventricle at normal resting potentials (negative
to -80 mv) is consistent with the absence of the
IK, system identified in Purkinje fibers.
A small inward transmembrane current (back-
ground current) may flow during ventricular phase
4 depolarization at maximum diastolic potentials
positive to -60 mv. This possibility is suggested by
the reduction of phase 4 slopes for maximum
diastolic potentials above -65 mv when either Ca
or Na was reduced (Fig. 5) and when tetrodotoxin
was used (Fig. 7). However, it is impossible to rule
out an indirect mechanism, i.e., modification of K
Circulation Research, Vol. 37, July 1975
axons (34) and thus lead to a decrease in phase 4
slope. Reduction of [NaL through an increase in
[Ca]j might increase K permeability as has been
reported for motoneurons (35). The inability of
elevated [Ca]o to accelerate phase 4 depolarization
in several preparations (Fig. 3, sections 8 and 9)
can probably be explained on the basis of an
increase in K permeability. The effect of [Ca]o on
K permeability is also suggested by the inverse
relationship which was consistently observed be-
tween [Ca]o and the duration of the regenerative
action potentials. This concept is supported by
evidence from other tissues of a direct relationship
between intracellular Ca concentration and K
permeability (35).
THE REGENERATIVE SPIKE PROCESS
The present results support the concept that
both Na and Ca participate in the regenerative
action potentials of ventricular automaticity. The
major evidence for the participation of Na is the
significant positive shift in threshold and the
decrease in upstroke velocity when Na is reduced in
the perfusate or when tetrodotoxin is added.
Changes in [Ca]o have relatively little effect on the
relationship between maximum diastolic potential
and threshold, but at less negative maximum
 126
diastolic potentials Ca depletion abolishes regener-
ative spikes before diastolic depolarization is elimi-
nated.
The reduction in action potential overshoot by
reduction of either [Ca]o or [Na]0 was a consistent
finding. This finding is similar to that reported for
conventional stimulated action potentials in frog
(17, 36) and guinea pig (37, 38) ventricles. How-
ever, the very marked effect of [Na]o reduction on
the occurrence of action potentials at less negative
potentials (Fig. 4, sections 6 and 9) was not
reproduced by high concentrations of tetrodotoxin
(Fig. 7, section 6). Thus, it appears likely that this
effect of [Na]o reduction might have resulted from
intracellular Ca accumulation (22, 23) and a result-
ing decrease in Ca current. Alternatively, the
possibility that tetrodotoxin-insensitive channels
for Na current exist in the guinea pig ventricle
must be considered. Evidence for such channels in
the chick embryo heart has been reported (39).
Ca clearly plays an important role in the genesis
of regenerative action potentials. It is well docu-
mented that Ca can support regenerative action
potentials in cardiac cells in Na-free solutions and
in K-depolarized or tetrodotoxin-treated prepara-
tions in which the Na conductance is presumably
completely inactivated or blocked (16, 21). In the
Downloaded from http://ahajournals.org by on July 29, 2020
present experiments, [Ca]o was directly related to
action potential upstroke dV/dt and to action
potential overshoot at all membrane potentials. In
addition, reduction of [Ca]o to 10% of normal
usually eliminated regenerative spiking at less
negative potentials.
These results suggest that depolarization-in-
duced automaticity in ventricular fibers is similar
to that in Purkinje fibers. The data do not permit a
definitive explanation of depolarization-induced
automaticity but emphasize the need for further
study of this phenomenon in a variety of cardiac
cells. Finally, it should be recognized that ade-
quate spatial control (space clamp) is important in
current clamps as well as in voltage clamps (21,
40). Nonhomogeneous distribution of current to
different portions of the tissue under study could
reproduce many of the results reported in the
present paper.
Addendum
Since this paper was submitted, two abstracts
have been published relating to depolarization-
induced automaticity in ventricular fibers (Suraw-
icz and Imanishi, Circulation 50:111-84, 1974, and
Imanishi and Surawicz, Circulation 50:111-145,
1974).
KATZUNG
Acknowledgment
I thank Dr. A. O. Grant and Dr. L. Hondeghem for useful
suggestions regarding this research and for a critical reading of
the manuscript. I thank Charles Lee and Charles Cotner for
technical assistance and Thelma Brunn for secretarial assist-
ance.
References
1. KATZUNG BG: Electrically induced automaticity in ventricu-
lar myocardium. Life Sci 14:1133-1140, 1974
2. KATZUNG BG: Drug effects on ventricular automaticity. Proc
West Pharmacol Soc 17:15-18, 1974
3. TRAUTWEIN W, KASSEBAUM DG: On the mechanism of
spontaneous impulse generation in the pacemaker of the
heart. J Gen Physiol 45:317-330, 1961
4. HAUSWIRTH 0, NOBLE D, TSIEN RW: Mechanism of oscilla-
tory activity at low membrane potentials in cardiac
Purkinje fibers. J Physiol (Lond) 200:255-265, 1969
5. IMANISHI S: Calcium-sensitive discharges in canine Purkinje
fibers. Jap J Physiol 21:443-463, 1971
6. SPERELAKIS N, LEHMKUHL D: Effect of current on transmem-
brane potentials in cultured chick heart cells. J Gen
Physiol 47:895-927, 1964
7. BROWN HF, NOBLE SJ: Membrane currents underlying
delayed rectification and pace-maker activity in frog
atrial muscle. J Physiol (Lond) 204:717-736, 1969
8. LENFANT J, MIRONNEAU J, A K A J - K : Activite repetitive de la
fibre sino-auiculaire de grenouille: Analyse des courants
membranaires responsables de l'automatisme cardiaque.
J Physiol (Paris) 64:5-18, 1972
9. TRAUTWEIN W: Membrane currents in cardiac muscle fibers.
Physiol Rev 53:793-835, 1973
10. NOBLE D, TSIEN RW: Repolarization process of heart cells.
In Electrical Phenomena in the Heart, edited by WC
DeMello. New York, Academic Press, 1972, pp 133-161
11. BROOKS C, LU H: The Sino-Atrial Pacemaker of the Heart.
Springfield, Illinois, Charles C Thomas, 1972
12. WEST TC: Electrophysiology of the sino-atrial node. In
Electrical Phenomena in the Heart, edited by WC De-
Mello. New York, Academic Press, 1972, pp 191-217
13. SPERELAKIS N, LEHMKUHL D: Ionic interconversion of pace-
maker and nonpacemaker cultured chick heart cells. J
Gen Physiol 49:867-895, 1966
14. DRAPER MH, WEIDMANN S: Cardiac resting and action
potentials recorded with an intracellular electrode. J
Physiol (Lond) 115:74-94, 1951
15. WEIDMANN S: Effects of calcium ions and local anesthetics
on electrical properties of Purkinje fibers. J Physiol
(Lond) 129:568-582, 1955
16. ARONSON RS, CRANEFIELD PF: Electrical activity of canine
cardiac Purkinje fibers in sodium-free, calcium-rich solu-
tions. J Gen Physiol 61:786-808, 1973
17. HAGIWARA S, NAKAJIMA S: Differences in Na and Ca spikes
as examined by.application of tetrodotoxin, procaine, and
manganese ions. J Gen Physiol 49:793-806, 1966
18. DUDEL J, PEPER K, RUDEL R, TRAUTWEIN W: Effect of
tetrodotoxin on the membrane current in cardiac muscle
(Purkinje fibers). Pfluegers Arch 295:213-226, 1967
19. REUTER H, SCHOLTZ H: Ober den Einfluss der
extracellularen Ca-Konzentration auf Membranpotential
und Kontraktion isolierter Herzpraparate bei graduierter
Depolarisation. Pfluegers Arch 300:87-107, 1968
20. HODGKIN AL: Ionic basis of electrical activity in nerve and
muscle. Biol Rev 26:339-409, 1951
Circulation Research, Vol. 37, July 1975
 Ca AND Na ON VENTRICULAR AUTOMATICITY
21. REUTER H: Divalent cations as charge carriers in excitable
membranes. In Progress in Biophysics and Molecular
Biology, vol 26, edited by JAV Butler and D Noble.
Oxford, Pergamon Press, 1973, pp 1-43
22. NIEDERGERKE R: Movements of Ca in frog heart ventricles at
rest and during activity. J Physiol (Lond) 167:515-550,
1963
23. GLITSCH H, REUTER H, SCHOLTZ H: Effect of the internal
sodium concentration on calcium fluxes in isolated
guinea-pig auricles. J Physiol (Lond) 209:25-43, 1970
24. ARONSON RS, CRANEFIELD PF: Effect of resting potential on
the electrical activity of canine cardiac Purkinje fibers
exposed to Na-free solution or to ouabain. Pfluegers Arch
347:101-116, 1974
25. ANTONI H, TAGTMEYER H: Die Wirkung starker Strome auf
Erregungsablauf und Kontraktion des Herzmuskels:
Beitrage zur Ersen Hilfe und Behandbung von Unfalle
Durch Elektrischen Strom. Frankfurt a.M., Verlags-
u. Wirtschaftsges. d. Elektrizitatswerke, 1965, p 38
26. WIT AL, ROSEN MR, HOFFMAN BF: Electrophysiology and
pharmacology of cardiac arrhythmias: II. Relationship of
normal and abnormal electrical activity of cardiac fibers
to the genesis of arrhythmias. Am Heart J 88:515-524,
1974
27. WEIDMANN S: Effect of current flow on the membrane
potential of cardiac muscle. J Physiol (Lond)
115:227-236, 1951
28. NOBLE D, TSIEN RW: Kinetics and rectifier properties of the
slow potassium current in cardiac Purkinje fibers. J
Physiol (Lond) 195:185-214, 1968
29. NOBLE D, TSIEN RW: Outward membrane currents acti-
vated in the plateau range of potentials in cardiac
Purkinje fibers. J Physiol (Lond) 200:205-231, 1969
Downloaded from http://ahajournals.org by on July 29, 2020
30. OJEDA C, ROUGIER O: Kinetic analysis of the delayed
Circulation Research, Vol. 37, July 1975
127
outward currents in frog atrium: Existence of two types of
preparation. J Physiol (Lond) 239:51-73, 1974
31. MCGUIGANJAS: Some limitations of the double sucrose gap,
and its use in a study of the slow outward current in
mammalian ventricular muscle. J Physiol (Lond)
240:775-806, 1974
32. OCHI R: Slow inward current and the action of manganese
ions in guinea-pig's myocardium. Pfluegers Arch
316:81-94, 1970
33. WALDEN M, KREHER P, AKA K-J, TRICOCHE R: Activite
electrique et courants ioniques transmembranaires de la
fibre myocardique de singe (famille des cercopithecides).
J Physiol (Paris) 66:455-472, 1973
34. FRANKENHAEUSER B, HODGKIN AL: Action of calcium on the
electrical properties of squid axons. J Physiol (Lond)
137:218-244, 1957
35. FELTZ A, KRNJEVIC K, LISIEWICZ A: Intracellular free Ca and
membrane properties of motoneurones. Nature [New
Biol] 237:179-181, 1972
36. NIEDERGERKE R, ORKAND RK: Dual effect of calcium on the
action potential of the frog's heart. J Physiol (Lond)
184:291T311, 1966
37. CORABOEUF E, OTSUKA M: L'action des solutions hyposo-
diques sur les potentiels cellulaires de tissu cardiaque de
Mammiferes. CR Acad Sci p ] (Paris) 243:441-444, 1956
38. CORABOEUF E, VASSORT G: Effects of some inhibitors of ionic
permeabilities on ventricular action potential and con-
traction of rat and guinea-pig hearts. J Electrocardiol
1:19-30, 1968
39. MCLEAN MJ, SHIGENOBU K, SPERELAKIS N: TWO pharmaco-
logical types of cardiac slow Na + channels as distin-
guished by verapamil. EurJ Pharmacol 26:379-382, 1974
40. TAYLOR RE, MOORE JW, COLEKS: Analysis of certain errors
in squid axon voltage clamp measurements. Biophys J
1:161-201, 1960