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Inotropic Effects of Different Calcium Ion Concentrations
on the Embryonic Chick Ventricle
COMPARISON OF SINGLE CULTURED CELLS AND INTACT MUSCLE STRIPS
By William H. Barry, Roger Pitzen, Kenneth Protas, and Donald C. Harrison
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ABSTRACT
The effects of changes in the calcium ion concentration in the medium bathing single
beating cultured chick embryo ventricular cells were determined using an electro-optical
monitoring technique for measuring the amplitude and velocity of cell wall motion.
Although single cells showed significant decreases in the amplitude and velocity of cell
wall motion during contraction when the calcium ion concentration was lowered from
1.8 mM to 0.9 raM, there were no significant changes in these parameters when the
concentration was increased from 1.8 mM to 3.6 mM. Ventricular strips obtained from
embryos of the same age showed positive inotropic responses to an increase in the external
calcium ion concentration from 1.8 mM to 3.6 mM, and the magnitude of this response
increased with increasing embryonic age. These results suggest that there is a reduced
positive inotropic responsiveness in young chick embryo ventricular cells, which is
particularly marked in the single cultured cell preparation because of the tendency of
culturing techniques to select out immature cell populations.
KEY WORDS
potassium concentration
contraction velocity
electro-optical monitoring
contractile force
• Cultured single cardiac cells offer significant
advantages for studying the action of pharmacologic agents. These denervated preparations are
stable at physiological temperatures, and electrooptical techniques (1-3) provide a means of monitoring the rate of contraction and the amplitude
and velocity of cell wall motion. Thus, the single
cultured cell preparation theoretically allows the
inotropic and chronotropic effects of drugs to be
studied.
Using this preparation, we have previously
shown that chronotropic effects of parasympathetic
and sympathetic drugs can be demonstrated in the
cultured chick embryo ventricular cell (1, 4, 5). In
the course of these studies, however, we noted that
isoproterenol, while inducing an increase in the
rate of contraction of these cells, has relatively
little effect on the amplitude or velocity of cell wall
motion. This observation suggests that our monitoring techniques do not detect inotropic effects.
However, studies by Okarma et al. (6), using
similar monitoring techniques, have demonstrated
a positive inotropic effect in the single cultured rat
From the Cardiology Division, Stanford University School of
Medicine, Stanford, California 94305.
This work was supported by U. S. Public Health Service
Grants HL-5866 and 1-P01-HL-15833-01 from the National
Heart and Lung Institute.
Received August 6, 1974. Accepted for publication March 6,
1975.
Circulation Research, Vol. 36, June 1975
cell wall motion
tissue culture
heart cell following exposure to digitalis glycosides,
and studies by Thompson et al. (7) have shown an
inotropic effect of norepinephrine in the single
cultured mouse heart cell using similar methods.
Thus, it appears that our techniques should be
capable of detecting inotropic changes which are
reflected in changes in cell wall contraction amplitude and velocity and that, perhaps, the cultured
chick ventricular cell is unresponsive to inotropic
agents.
The inotropic responsiveness of the chick embryo
ventricle of various ages has been studied previously by a number of investigators, whose results
have been varied. In general, earlier studies showed
a positive inotropic effect in response to an increase
in the calcium ion concentration bathing the tissue
(8) and an inotropic effect due to exposure to
catecholamines (9, 10). However, recent studies by
Shigenobu and Sperelakis (11) suggested that the
myocardium from 5- and 4-day-old embryos is
insensitive to the inotropic effect of catecholamines, whereas the myocardium from older embryos (8-10 days of age) is responsive.
Preliminary studies done in our laboratory (12)
have suggested a significant increase in inotropic
responsiveness with an increase in the age of the
embryo when isolated muscle strips are studied
between the age of 4 and 12 days. The inotropic
responsiveness to elevations in external calcium ion
727
728
BARRY. PITZEN. PROTAS. HARRISON
concentration and that to exposure to isoproterenol
are similar. Therefore, the present study was designed to determine the effect of a simple inotropic
agent, calcium ion, on the amplitude and velocity
of cell wall contraction of a cultured single cell
preparation obtained from a chick embryonic ventricle and to compare the inotropic response to
calcium of the single cell with that seen in strips of
ventricular myocardium of the same age. The
purpose of this study was to determine whether the
cultured single embryonic ventricular cell can be
used for studying the effects of inotropic agents; we
also wanted to know if the cultured cell differs from
the intact muscle strip and if relative inotropic
unresponsiveness does indeed occur in younger
embryonic tissue.
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Methods
CULTURED SINGLE CELL PREPARATION
Cell Culturing.—Cell cultures were prepared with
some minor modifications (5) according to the DeHann
method (13). Six to eight chick embryos were aseptically
removed from 6-day-old specific pathogen-free eggs (LegWhitehorn), the ventricles were dissected out, and ventricular fragments were dispersed in 0.05% trypsin.
Washed trypsin-dispersed cells were resuspended in 2 ml
of the final maintenance medium, which consisted of
20% M 199 nutrient medium, 6% heat-inactivated fetal
calf serum, 1% penicillin-streptomycin antibiotic solution, and 73% potassium-free balanced salt solution. The
cell suspension was poured through sterile bolting silk to
remove undissociated cell clumps, counted on a hemocytometer, and diluted to give a stock cell suspension of 4 x
105 cells/ml maintenance medium. The cells were then
seeded into Sykes-Moore chambers, which were assembled immediately prior to use from autoclaved components. An aliquot of 0.25 ml of the stock cell suspension
was transferred into each chamber with a 1-ml sterile
pipette, the top coverslips were put into place, and the
upper ring was secured. Each chamber therefore received
1 x 106 cells. The cultures were incubated in a humidified 95% air-5% CO2 atmosphere at 37°C. Within 48
hours the cells became attached to the glass, and within
48-72 hours the cells were observed and recordings of the
spontaneously beating cells were begun. In these cultures, approximately 60% of the attached cultured cells
showed spontaneous contractile activity.
Recording Technique.—Experimental
observation
and recording of the heart cell motion were accomplished
using a previously described (1,5) electro-optical system
that was developed as a noninvasive method of monitoring cultured heart cell activity. Briefly, the Sykes-Moore
chambers containing the heart cells to be monitored were
placed in a machined frame designed to prevent spurious
movement and suspended over the aperture of the
movable stage of an inverted Wild phase-contrast microscope surrounded by a Plexiglas incubator. A humidified
95% air-5% CO2 atmosphere was maintained at 37°C by a
temperature-sensitive probe and a heater apparatus. The
image of a single cell was viewed from a side-arm adapter
of the microscope by a GBC-ITC transistorized television
camera (model CTC 5000) and cabled to a Conrac
television monitor. The total magnification of the cell
image on the television screen was 1400x. A photovoltaic
cell with a rectangular aperture dimension of 10 x 20 mm
was positioned on the television screen over one edge of
the single beating heart cell wall in such a way that the
wall motion resulted in a proportionate voltage output.
As the cell contracted and relaxed, the light intensity in
the field of the photocell was altered, causing a change in
voltage. This signal, corresponding to the amplitude of
cell wall motion, and its first derivative, corresponding to
cell wall velocity, were recorded using a Krohn-Hite filter
(band pass 0-20 Hz) and a Beckman-Offner direct-writing recorder-amplifier system. Such a system is capable
of determining the rate of beating and the relative cell
wall motion—cell wall amplitude and velocity. Our
primary concern was changes in these parameters following interventions; since cell wall motion varied with
position and portion of the cell monitored, only relative
amplitude and velocity of the cell wall were measured,
keeping the position of the photocell and the cell wall
constant.
Three 22-gauge needles were inserted obliquely into
the culture chambers through three of the ports of the
chamber frame. Inserted obliquely through the fourth
port was a 20-gauge needle which acted as an air vent.
The needles, with the exception of the air vent, were
positioned in the chamber in such a way that the bevel of
each needle was parallel to and resting on the bottom
coverslip. Attached to each of these needles were 50-cm
lengths of polyethylene tubing which were in turn
connected to syringes fastened to the interior wall of the
incubator. The barrels of the syringes extended outside
the incubator through small holes which had been drilled
into the wall, thereby permitting the manipulation of the
syringes from outside the incubator while keeping the
media within the syringes at 37°C. Using this system, the
medium could be completely removed from the culture
chamber with one aspiration of an empty 10-ml syringe
and immediately replaced with an equal volume of
medium from a 1-ml syringe. Using this method, medium changes were accomplished within 30 seconds
without moving the cell in the microscope stage.
Experimental Procedure.—Only one cell per culture
chamber was monitored during each study. Prior to
beginning a study, the maintenance medium containing
1.8 mM calcium in the culture chamber was withdrawn
and replaced with 0.25 ml of fresh warm 1.8 mM calcium
maintenance medium. The chamber was allowed to sit
on the stage of the microscope for at least 40 minutes,
enabling the cells to adjust to the medium change.
During this equilibration period, an attempt was made to
locate a stable beating cell that was regular in rate and
relatively constant in amplitude of contraction. When a
cell was chosen, occasional recordings were made until
the 40-minute equilibration period was over to establish
that the cell was stable. A series of medium changes was
then performed according to the following procedure. At
the end of the 40-minute stabilization period, the maintenance medium containing 1.8 mM calcium was withdrawn and 0.25 ml of maintenance medium with the
same calcium concentration was added. Starting immediately after the medium change, cell wall amplitude
and velocity and the rate of beating were monitored for
20 seconds each minute for the next 10 minutes. At
Circulation Research, Vol. 36, June 1975
729
CALCIUM EFFECTS ON CULTURED VENTRICULAR CELLS
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10-minute intervals, the medium was changed to a
medium containing a different calcium concentration.
Three different calcium concentrations were studied in
the following sequence: 1.8 mM, 0.9 mM, 1.8 IDM, 3.6 mM,
1.8 mM. A total of 15 cultured cells from 6-day-old
embryos was studied, 7 in the presence of low potassium
concentration (1.8 mM, identical to that in the culture
solution) and 8 in the presence of a normal potassium
concentration (4.0 mM). In addition, 7 cells were studied
in cultures obtained from 10-day-old embryos (4.0 mM
potassium). The rate of beating for a given minute was
calculated by counting the number of contractions that
occurred during a 15-second period. To determine the
relative amplitude and velocity of contraction, ten primary and differentiated contraction signals were measured and averaged to yield an average relative amplitude and velocity of contraction for that time period.
These values were then used in statistical analysis of the
data. The amplitude and velocity of contraction of each
cell at the end of the 40-minute stabilization period were
taken as the 100% values for normalization. Only cells
that continued to beat during the entire study period were
included in the analysis.
ISOLATED MUSCLE STRIP PREPARATION
Hearts were removed from their embryos and placed in
Tyrode's solution bubbled with 95% O2-5% CO2. The
ionic composition of this solution was identical to that of
the cultured heart cell normal potassium solution, except
for the presence of 6% fetal calf serum in the latter, and
contained NaCl 116 mM, NaH2PO4 • H2O 0.9 mM,
MgSO4 • 7 H2O 0.8 mM, NaHCO3 26.2 mM, CaCl2 • 2 H2O
1.8 IBM, KC1 4.0 mM, and dextrose 5.5 mM. A thin strip of
ventricular muscle 0.4-0.8 mm in diameter and 2-3 mm
in length was dissected free, using a Wild dissecting
microscope and a sharp scalpel. This muscle strip was
mounted in a muscle bath perfusion chamber with 3/0
silk thread loops passed through the ends of small glass
capillary tubes. One capillary tube was attached to an
Imperial Controls DSC microforce transducer, and the
other was attached to a micromanipulator. This latter
tube contained a small platinum wire for muscle stimulation. The perfusion chamber was perfused with oxygenated heated solution bubbled with 95% O2-5% CO2,
and the temperature was maintained at 37°C. The
perfusion chamber had a volume of 3 ml, which could be
emptied by suction and refilled by gravity with fresh
perfusion fluid containing various concentrations of calcium ions within 30 seconds. After dissection and mounting, the strips were allowed to equilibrate without
stimulation and without resting tension for 40 minutes in
the muscle bath. At the end of this 40-minute period,
stimulation was begun at 100/min; the strips were
stretched to the peak of their isometric length-tension
curve, and the length was then reduced back to the point
at which they were producing half of the peak maximum
contractile force. This length was maintained constant
throughout the remainder of the study. Using this
technique, stable preparations were obtained from hearts
of 4-, 6-, and 8-day-old embryos. The initial perfusion
chamber solution change was performed maintaining the
calcium concentration at the normal level of 1.8 mM; this
step served as a control. Subsequently, every 10 minutes
the perfusion chamber was drained and refilled with a
medium of a different calcium concentration in the same
order as that used in the cultured 6-day-old heart cell
Circulation Research, Vol. 36, June 1975
study. Peak forces were measured during contraction at
1-minute intervals. During these studies, the muscle
strips were stimulated with a square wave 4 msec in
duration and 25% over threshold delivered from a Tektronic pulse generator and a WPI stimulus isolation unit.
The electrodes were positioned so that the strips could be
stimulated when the muscle bath was completely
drained to prevent cessation of stimulation during solution changes. Between emptyings of the perfusion chamber, the chamber was perfused constantly with the
appropriate solution at a rate of 5 ml/min. The contractile force was recorded using an a-c-coupled BeckmanOffner recorder-amplifier system that was linear from 0.4
to 20 Hz.
Results
CULTURED SINGLE CELL PREPARATION
Satisfactory recordings were obtained from 15
single ventricular cells cultured from 6-day-old
embryos. An example of one such recording is
shown in Figure 1". On lowering the calcium concentration from 1.8 mM to 0.9 mM, there was a
dramatic and rapid decrease in the amplitude and
velocity of cell wall motion. However, following an
increase in the calcium concentration from 1.8 mM
to 3.6 mM, there was relatively little change in
these parameters. Similar studies were done in 8
cells in the presence of a normal potassium concentration of 4.0 mM and in 7 cells in the presence of
1.8 mM potassium, which was the standard concentration of potassium in the maintenance culture
medium. Results in these two groups did not differ
and were combined for presentation. These results
are shown for the 15 cells in Figure 2. It is apparent
that for the entire group of cells the relative cell
wall amplitude and relative cell wall velocity fell
significantly when the calcium concentration was
lowered from 1.8 mM to 0.9 mM but did not increase
significantly when the calcium concentration was
raised from 1.8 mM to 3.6 mM. It is also apparent
that changes in amplitude and velocity occurred
quite rapidly with changes in calcium concentration.
Changes in the rate of contraction for these
various calcium concentrations were also noted. All
medium changes, including the control change,
induced a transitory 10-20% increase in the rate of
contraction which lasted 3-4 minutes. We presume
that this transient increase in the rate of contraction resulted from a mechanical effect of the
medium change (14). However, there were no
significant differences in the rate of contraction at
the end of each 10-minute period following a
solution change (~110/min).
In eight cells obtained from cultures of 10-dayold embryo ventricles, the effect of elevating the
calcium concentration was also investigated. These
730
BARRY, PITZEN. PROTAS. HARRISON
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FIGURE 1
Records demonstrating the effects of changes in external calcium ion concentration ([Ca++]) on the
contractile rate, cell wall amplitude (top trace) and cell wall velocity (bottom trace) of a single cultured cell obtained from a 6-day-old embryo. The cell had equilibrated in fresh medium containing
1.8 m*A calcium for 40 minutes before the first medium change. The times when the bathing medium
was removed and replaced are shown by vertical arrows. The external calcium concentrations are
shown above the records. Following each medium change, recordings were collected for 10 minutes.
results are shown in Figure 3. It can be seen that for
the hearts from the older embryos there was no
significant increase in the relative cell wall motion
amplitude during contraction when the calcium
concentration bathing the cells was raised from 1.8
mM to 3.6 mM.
ISOLATED MUSCLE STRIP PREPARATION
Figure 4 shows an example of the data obtained
with a 6-day-old ventricular strip in the perfused
muscle bath. Contractile force did not change
significantly when the perfusion chamber was emptied and filled and calcium concentration was kept
constant (control). When the calcium concentration bathing the strip was lowered from 1.8 mM to
0.9 mM, there was a decrease in contractile force,
which returned to control levels when the calcium
concentration was increased to 1.8 mM. When the
calcium concentration was increased to 3.6 mM,
there was a highly significant increase in contractile force. Figure 5 shows the data obtained from
eight 6-day-old embryo ventricular strips. The
Circulation Research. Vol. 36. June 1975
731
CALCIUM EFFECTS ON CULTURED VENTRICULAR CELLS
the younger embryos had a blunted increase in
contractile force on exposure to high calcium concentrations compared with that in older hearts.
However, ventricular strips from 6-day-old embryos showed a definite and significant increase in
contractile force when they were exposed to high
calcium concentrations, whereas cultured cells
from embryos of the same age did not.
FIGURE 2
Plot of relative cell wall amplitude (top) and relative cell wall
velocity (bottom) against time for 15 single cells cultured from
6-day-old chick embryos. The points indicate the means and the
brackets the SE. The cells had equilibrated for 40 minutes before
the first medium change, which was for control purposes. The
calcium concentration (mM) in each medium is indicated above
the vertical arrows, which indicate the time of the changes.
Comparison by paired t-test of amplitude of contraction at
various times yielded the following: 50 vs. 60 minutes P < 0.025,
60 vs. 70 minutes P < 0.01, 70 vs. 80 minutes NS, 80 vs. 90
minutes NS, 50 vs. 70 minutes NS, 70 vs. 90 minutes NS, 50 vs. 90
minutes NS. Paired t-test analysis of velocity of contraction at
various times yielded: 50 vs. 60 minutes P < 0.01, 60 vs. 70
minutes P < 0.001, 70 vs. 80 minutes NS, 80 vs. 90 minutes NS, 50
vs. 70 minutes NS, 70 vs. 90 minutes NS, 50 us. 90 minutes NS.
decrease in contractile force in the muscle strip
preparations on going from 1.8 mM to 0.9 mM calcium was more marked than the change in the
single cultured cells from embryo hearts of the same
age and had a more gradual course. In addition,
when the calcium concentration was increased
from 1.8 mM to 3.6 mM, there was a very significant
increase in contractile force. It should also be noted
that the time constant of the decrease in contractile force following the lowering of the calcium concentration was somewhat greater (2.4 minutes)
than the time constant of the increase in contractile force (1.5 minutes) following the increase in
calcium concentration bathing the muscle strips.
Similar data were obtained from hearts of 4- and
8-day-old embryos; the percent increase in contractile force is plotted against embryo age in days
in Figure 6. The percent increase in contractile
force following an increase in external calcium
concentration from 1.8 mM to 3.6 mM increased
significantly with increasing embryonic age. Thus,
Circulation Research, Vol. 36. June 1975
AMPLITUD
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60
70
TIME (minutes)
Discussion
These studies demonstrated that cultured single
ventricular cells from 6- and 10-day-old embryonic
ventricles are insensitive to the inotropic effects of
increasing the external calcium concentration from
1.8 mM to 3.6 mM. However, these cells do reduce
their amplitude and velocity of contraction when
they are exposed to lower external calcium concentrations. These findings suggest that these cells are
operating at their maximum inotropic level in the
presence of a calcium ion concentration of 1.8 mM.
It should be noted that in most mammalian serums
and in 20-day-old chick embryo serum, the normal
ionized calcium concentration is 0.9-1.1 mM. We
were not able to obtain enough serum from embryos of the ages studied in these experiments to
ascertain this normal ionized calcium concentration; however, we assume it is about 0.9 mM. The
presence of 6% fetal calf serum in the solutions used
in the cultured cell studies did not significantly
alter the total ionized calcium levels, which were
essentially equal to total calcium concentrations.
In contrast to single cultured cells, ventricular
strips from 6-day-old embryos showed a significant
increase in contractile force during exposure to 3.6
1.8
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1.8
1
50
60
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TIME (minutes)
FIGURE 3
Plot of average relative cell wall amplitude against time during
external medium changes for eight single cells obtained from
10-day-old embryos. The time of each medium change is indicated by a vertical arrow, and the calcium concentration (mM)
in the new medium is shown above each arrow. There was no
significant change in amplitude with elevation of external
calcium to 3.6 mM.
BARRY. PITZEN. PROTAS. HARRISON
732
10
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FIGURE 4
Records showing the effect of changes in external calcium (Ca) ion concentration on contractile force
developed by a ventricular strip from a 6-day-old chick embryo. The strip had equilibrated for 40
minutes before the first (control) solution change. The point of each change is indicated by a vertical
arrow, the calcium concentration (mM) of the new solution is shown above the arrow. Ten minutes
elapsed between each calcium concentration change. The top trace in each record is a time marker
(seconds and minutes).
mM calcium solutions. In addition, in ventricular
strips there was a significant increase in the response to the positive inotropic effects of calcium
with increasing embryonic age. These findings at
37°C confirm our previous observations obtained at
lower temperatures (12) and are consistent with the
observations made by Shigenobu and Sperelakis
(11).
It is not clear at the present time why there is a
blunted inotropic response in the cultured cells. An
increase in inotropic responsiveness with increasing
age of embryonic tissue has been noted in human
myocardial tissue as well (15) and may be a
common phenomenon during fetal development.
The ventricular myocardium in an embryo of a
specific age contains cells of various degrees of
maturity. Since more immature cells generally
grow more readily in tissue culture, the single
cultured ventricular cell may be relatively immature and thus have a low inotropic responsiveness.
This explanation would account for our observation
that the inotropic response to an elevation in the
external calcium concentration in 6-day-old em-
bryonic muscle strips was more marked than that
in cultured single cell preparations obtained from
embryonic ventricles of the same age. The fact that
the cultured cells from 10-day-old embryonic ventricles also showed a lack of inotropic responsiveness suggests that, regardless of the age of the ventricle cultured, the resulting single beating cell
population is relatively uniformly immature. Thus,
although an increase in inotropic responsiveness
occurs with increasing embryonic age in muscle
strip preparations, the response of the single cultured cell obtained from older tissue remains
immature.
Lack of inotropic response to elevations in external calcium ion concentration above 1.8 mM could
mean that (1) extra calcium ions are not gaining
entrance to the cell in spite of the elevation of the
external calcium levels or (2) the myofibrils are not
developing more force in spite of the increased
calcium influx into cells during depolarization in
the presence of higher calcium concentrations. The
former situation could occur if a calcium-binding
or carrier element in the plasma membrane of these
Circulation Research, Vol. 36, June 1975
CALCIUM EFFECTS ON CULTURED VENTRICULAR CELLS
60
70
TIME (minutis)
FIGURE 5
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Plot of contractile force/area against time during changes in external calcium ion concentration for nine ventricular strips obtained from 6-day-old embryos. The circles indicate the means
and the brackets the SE. The vertical arrows indicate the times
of calcium concentration change, and the calcium concentration
(mM) is shown above each arrow. The strips had equilibrated for
40 minutes before the initial (control) change. Paired t-test
analysis at various times showed: 50 vs. 60 minutes P < 0.001,
60 vs. 70 minutes P < 0.001, 70 vs. 80 minutes P < 0.001, 80 vs.
90 minutes P < 0.001, 50 vs. 70 minutes NS, 70 vs. 90 minutes
NS, 90 vs. 50 minutes NS, 40 vs. 50 minutes NS.
young cells were saturated in the presence of 1.8
mM calcium. Sperelakis and Shigenobu (16) have
shown that ventricular cells in young embryos are
also insensitive to the effects of tetrodotoxin, which
blocks the early sodium current during the action
potential, whereas in older embryos tetrodotoxin
has its usual effect on the ventricular membrane
potential during depolarization. It is interesting
that this effect of tetrodotoxin on heart cells from
older ventricles disappears if the cells are cultured:
the immature type of response to tetrodotoxin is
seen in cultured cells from embryos of all ages (17).
EMBRYO AGE , doys
FIGURE 6
Plot of the percent increase in contractile force/area on elevation
of external calcium concentration from 1.8 to 3.6 mM for strips
from 4-, 6-, and 8-day-old embryos. By nonpaired t-test, there
were significant differences between 4- and 6-day-old embryo
strips (P < 0.05) and between 6- and 8-day-old embryo strips
Circulation Research, Vol. 36, June 1975
733
This observation is also consistent with the fact
that culture techniques may be selecting out a more
immature cell group. Thus, it is possible that
changes in membrane properties of chick embryonic hearts during development account for the
different responses to elevations in external calcium ion concentration noted. However, there is no
evidence that inotropic agents do not induce an
increase in the slow calcium ion currents in the
young embryonic chick ventricle (11).
Another possibility is that, although extra calcium is gaining entrance to the cells during depolarization in the presence of higher external calcium concentrations, the myofibrils are already
maximally activated by calcium during depolarization in the presence of normal external calcium
concentrations. This situation could occur if the
density of myofibrils were low, for the density of
myofibrils within a cell is a major determinant of
the amount of calcium ion required for full activation (18). If a constant amount of calcium ions
gains entrance to cells in the presence of a given
calcium concentration in the external medium,
more complete activation of the myofibrils could
occur in cells which have fewer myofibrils per cell.
Since the density of myofibrils increases during
embryonic development (19), this phenomenon
might also explain these observations. The observation by Ozaki (8) that monolayers of cultured
chick ventricle cells do show a positive inotropic
response to an elevation in the external calcium ion
concentration above 1.8 mM suggests that maturation of cells in culture also leads to increased
inotropic responsiveness. We cannot measure inotropic responses of monolayers of cells with our
technique. In any case, it would appear that the
cultured single embryonic chick ventricle cell is not
a good preparation to use to study the positive
inotropic effects of drugs in the presence of 1.8 mM
calcium, a concentration commonly used in tissue
culture, since most inotropic drugs are believed to
act by increasing the availability of calcium to
myofibrils during depolarization.
It is interesting to note that the time course of
change in cell wall contractile amplitude and
velocity following a reduction of calcium concentration in the cultured cell preparation is much
more rapid than the time course of change in
contractile force in the intact ventricular strip
(Figs. 2 and 5). This difference occurred in spite of
the facts that the muscle strips being studied were
extremely small in cross-sectional area and that
the cardiac tissue in these embryonic hearts has
relatively sparse connective tissue and might be
734
BARRY. PITZEN. PROTAS. HARRISON
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expected to be more readily permeable by external
diffusion. These findings suggest that the rate of
movement of calcium into muscle and out of cells is
diffusion limited in intact ventricular strips. This
finding and the observation that the time course of
change in contractile force in muscle strips during
an increase in external calcium concentration appears to be faster than that during a decrease in
external calcium concentration are consistent with
the observations of Toll and Jewell (20).
Langer and Frank (21) have shown that the
half-time for calcium washout from the perfused
rabbit heart septum is 1.4 minutes, whereas the
half-time for washout from a monolayer of cultured
rat heart cells is 1.15 minutes. The very rapid
changes in cell wall contraction amplitude and
velocity on lowering the external calcium concentration noted in our experiments suggest that the
single chick heart cell preparation has a calcium
washout half-time that is even shorter than that for
the monolayer of the cultured rat heart cell preparation in which calcium kinetics can be studied.
This difference might be due to the facts that some
interstitial space is present in a monolayer and that
the chick heart cells lack a T-tubule system.
Further studies are needed to define the anatomical and electrophysiological factors that underlie
the relative inotropic unresponsiveness of the immature chick embryo ventricular cell and the
development of such responsiveness in older tissues. Such studies may provide increased insight
into the mechanism of excitation-contraction coupling in normal mammalian cardiac muscle. In
addition, these studies emphasize the point made
by Rolett (22) that immaturity of heart cells in
tissue culture may be a factor of importance in
delineating cell responses to pharmacologic agents.
single beating heart cells in culture. Exp Cell Res
69:128-134, 1971
4. HARRISON DC, KLEIGER RE, MERIGA.N TC: Action of iso-
proterenol on heart cells in tissue culture. Proc Soc Exp
Biol Med 124:122-126, 1967
5. SINCLAIR AJ, BARRY WH, HARRISON DC: Effect of acetylcho-
line on single embryonic ventricle cells. Proc Soc Exp Biol
Med 139:165-168, 1972
6. OKARMA TB, TRAMELL P, KALMAN SM: Surface interaction
between digoxin and cultured heart cells. J Pharmacol
Exp Ther 183:559-576, 1972
7. THOMPSON EJ, WILSON SH, SCHUETTE WH, WHITEHOUSE WC,
NIRENBERG MW: Measurement of the rate and velocity of
movement by single heart cells in culture. Am J Cardiol
32:162-166, 1973
8. OZAKI S: Action of calcium on electrical and mechanical
activities of the cultured chick embryonic heart. Jap J
Physiol 19:632-640, 1969
9. MCCARTY LP, LEE WC, SHIDEMAN FE: Measurement of the
inotropic effects of drugs on the innervated and noninnervated embryonic chick heart. J Pharmacol Exp Ther
129:315-321, 1960
10. MICHAL F, EMMETT F, THORP RH: Study of drug action on
the developing avian cardiac muscle. Comp Biochem
Physiol 22:563-570, 1967
11. SHIGENOBL1 K, SPERELAKIS N: Calcium current channels
induced by catecholamines in chick embryonic hearts
whose fast sodium channels are blocked by tetrodotoxin
or elevated potassium. Circ Res 31:932-952, 1972
12. ADAMS MH, BARRY WH, HARRISON DC: Changes in inotropic
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responsiveness with increasing age of chick embryo ventricle (abstr). Circulation 46(suppl II): 11-138, 1972
DEHANN RL: Regulation of spontaneous activity and growth
of embryonic chick heart cells in tissue culture. Dev Biol
16:216-249, 1967
HASTEN FH: Automated single rose chamber for cardiac
cells. In Tissue Culture Methods and Applications, edited
by A Kruse and A Patterson. New York, Academic Press,
1973, pp 291-297
COLTART DJ, SPILKER BA: Development of human foetal
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525-526, 1972
SPERELAKIS N, SHIGENOBL' K: Changes in membrane properties of chick embryonic hearts during development. J Gen
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17. SHIGENOBL' K, SPERELAKIS N: Development of sensitivity to
Acknowledgment
The authors thank Dr. William Kivett for his assistance in the
early stages of this work, and Dr. Henry D. Schwartz for
performing the ionized calcium ion concentration determinations.
18.
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Circulation Research, Vol. 36. June 1975
Inotropic effects of different calcium ion concentration on the embryonic chick ventricle.
Comparison of single cultured cells and intact muscle strips.
W H Barry, R Pitzen, K Protas and D C Harrison
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Circ Res. 1975;36:727-734
doi: 10.1161/01.RES.36.6.727
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