472
11.
12.
13.
14.
15.
CIRCULATION RESEARCH
and Evans blue; evidence for increased vascular permeability in oedema and infection. Clin Sci 17: 639-646, 1958
Park MK, Baum D, Guntheroth WG: Lack of pooling in portal vein in
endotoxin shock in the dog. J Appl Physiol 28: 156-161, 1970
Guntheroth WG, Felsenfeld AJ: Splenic constriction with endotoxin
shock in the dog. J Appl Physiol 30: 517-520, 1971
Guntheroth WG, Hougen T, Kaplan EL: Absence of pooling with
endotoxin shock in the canine kidney. J Appl Physiol 32: 512-515,
1972
Chien S, Chang C, Dellenback RJ, Usami S, Gregersen MI: Hemodynamic changes in endotoxin shock. Am J Physiol 210: 1401-1410,
1966
Grannis FW: Guido Banti's hypothesis and its impact on the under-
16.
17.
18.
19.
20.
VOL. 41, No. 4, OCTOBER 1977
standing and treatment of portal hypertension. Mayo Clin Proc 50:
41-48,1975
Gilbert RP: Endotoxin shock in the primate. Proc Soc Exp Biol Med
111:328-331, 1962
Guntheroth WG, Mullins GL: Liver and spleen as venous reservoirs.
Am J Physiol 204: 3 5 ^ 1 , 1963
Fung YC: Theoretical considerations of the elasticity of red cells and
small blood vessels. Fed Proc 25: 1761-1772, 1966
Weideman MP: Dimensions of blood vessels from distributing artery
to collecting vein. Circ Res 12: 375-379, 1963
Chien S, Sinclair DG, Dellenback RJ, Chang C, Peric B, Usami S,
Gregersen MI: Effect of endotoxin on capillary permeability to macromolecules. Am J Physiol 207: 518-522, 1964
Mechanism of Activation of Contraction in Frog
Ventricular Muscle
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THOMAS W. ANDERSON, CARL HIRSCH, AND FREDERIC KAVALER
SUMMARY The force of contraction and transmembrane potential were recorded from extremely short segments
of frog ventricle strips, in which extracellular ionic composition could be changed rapidly, and a condition in which
there was a relatively uniform space clamp. Large and abrupt increases in contractile force (and, presumably, in
intracellular free calcium concentration) had no detectable effect on subsequent contractions. In contrast, an increase
in extracellular calcium concentration was associated with a progressive increase in contraction extending over several
beats. Displacement of transmembrane potential during an action potential affected contraction in a manner opposite
to that to be expected if a calcium current were to play a significant role in activation. The activation process could be
saturated by sufficient displacement of transmembrane potential to high inside positive levels. The voltage displacement necessary for saturation was less the higher the extracellular calcium concentration. Force development
occurring after displacement of transmembrane potential to levels beyond the equilibrium potential for calcium was
rapidly sensitive to alterations in extracellular composition, including addition of 10 mti Mn 2+ , an increase in calcium
concentration, and a decrease in sodium concentration. These observations provide further evidence that the source
of calcium for activation of contraction in frog ventricular muscle consists exclusively, or almost exclusively, of an
influx of this ion into the cell. This process contributes throughout the range of voltage dependence of activation: i.e.,
from threshold to saturation. The observations suggest that such transsarcolemmal calcium movement is brought
about by driving forces which add to those of electrodiffusion.
FROG VENTRICLE does not show a variety of positive
and negative inotropic effects of one beat on subsequent
ones, such as postextrasystolic potentiation and the weakness of the premature beat.1"4 This is in marked contrast to
mammalian cardiac muscle, in which such delayed inotropic effects are of great magnitude5"7 and have been
linked, on several grounds,4-*"10 to the function of intracellular calcium stores in the activation of contraction.
Intracellular bound calcium, which occurs in large
amounts in frog ventricular muscle," thus may not play an
important role in activation, a view also in accord with
From the Department of Physiology, Downstate Medical Center, State
University of New York, Brooklyn, New York.
Supported by Grant HL12774 from the National Heart and Lung
Institute. National Institutes of Health. Dr. Anderson was the recipient of
a New York Heart Association Fellowship during the period when these
experiments were conducted.
Dr. Hirsch's present address is: Department of Biology, Queens College. Flushing, New York.
Address for reprints: Dr. Thomas W. Anderson, New York Veterans
Administration Hospital. First Avenue at East 24th Street. New York.
New York 10010.
Received November 17.1975; accepted for publication March 9, 1977.
several reports that sarcoplasmic reticulum is sparse and T
tubules absent in this tissue.12"14
Other aspects of the behavior of frog ventricle appear,
in contrast, to be most easily understood in terms of a
significant contribution of calcium storage sites to contraction. These include staircase and the frequency-force relationship15 and a prolonged, progressive effect on contraction following alteration of extracellular calcium concentration and occurring over many beats. 12 '' 6 In addition,
the positive inotropic action of caffeine has been interpreted in this way and expecially the observation of caffeine-induced force development by the frog atrial trabecula in an essentially calcium-free medium.17
The contribution of calcium entry, during the action
potential, to the activation of contraction (in turtle ventricle) is most strongly indicated by the fact that an increase
in extracellular calcium concentration, initiated after the
onset of a contraction, increases the force of the same
beat.18 In addition, in frog ventricle, premature relaxation
rapidly follows a prematurely induced repolarization2'l9-20
or a sudden reduction in extracellular calcium concentration induced during a prolonged voltage clamp pulse.21
ACTIVATION OF CONTRACTION IN FROG VENTRICLE/Anderson el al.
In the experiments to be reported, the combined techniques of voltage clamp and of rapidly induced alterations
in extracellular composition were used to assess further
the relative contributions of transmembrane calcium entry
and of intracellular calcium release to the activation of
contraction in frog ventricular muscle. The observations
reported offer further evidence that the source of calcium
which mediates activation consists exclusively, or almost
exclusively, of an influx into the cell and give support to a
suggestion20-22 that such an influx may arise from other
than purely electrodiffusional driving forces. That the calcium sink (intracellular binding or extrusion) may be an
important additional determinant of activation is not excluded by these findings (see Figure 9 and Discussion).
Methods
PREPARATION
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Strips of frog ventricle (Rana pipiens) were dissected
from muscle running parallel to the atrioventricular
groove. The experimental arrangement is shown in Figure
1. Each strip, 0.8-1.0 mm wide, was mounted by drawing
its slightly narrowed, conical end through a hole in a
rubber partition until the Rt was fairly snug. The hole was
then reduced in size by partially releasing the tension on
the rubber sheet. The muscle was firmly snared by a silk
thread which connected it, through a rigid glass tube, to an
RCA 5734 mechanoelectric transducer, and the end of the
muscle was cut off close to the snare. A short segment of
the strip thus extended forward, from the rubber partition
to the snare, for a distance of 0.10-0.14 mm (at unstretched length). A much longer segment (over 4.0 mm)
extended back behind the partition and floated free in a
FIGURE 1 Diagram of the experimental arrangement for recording contraction from a very short segment (about 0.12 mm, at slack
length) of a strip of frog ventricle, during rapid alteration in
composition of the extracellular fluid. The muscle is held tightly by
a hole in a rubber partition and by a silk snare which secures the
forward end of the short segment to a force transducer. Most of the
strip (long segment) extends backward from the hole in the rubber
partition, floating unrestrained in a Ringer-filled channel in a
Perspex block (cross-hatched structure). The rubber is applied
under tension to the forward face of the Perspex. Also shown, on
the right: the provision for recording transmembrane potential and
for either its feedback control, in voltage clamp mode, or the
eliciting of ordinary action potentials, in stimulus mode. See text
for details.
473
Ringer-perfused cylindrical channel. A jet of Ringer's
solution played vigorously on the short segment, surrounding it with a cuff of fluid which ran off continuously
to the floor of the chamber. Flow over each of the segments was at a rate of 10 ml/min, or rapid enough to
replace the fluid around the segments more than 10 times
each second. The short segment was adjusted to a length
just under that optimal for contraction. In satisfactory
preparations, the contraction recorded was not affected by
changes in calcium concentration in the solution superfusing the long segment, and sodium-lack contracture in this
portion of the strip was not transmitted to the force transducer.
ELECTRICAL RECORDING AND STIMULATION
Transmembrane potential was led off from intra- and
extracellular, KCl-filled, glass microelectrodes, whose tips
were very close to one another at about 3/io of the distance, along the short segment, from the rubber partition
to the silk snare. This potential difference, together with
an appropriate bias voltage and command signals, was
applied to the summing junction of an operational amplifier. Through a pushbutton switch, either the feedback
amplifier output or a 5-msec stimulus pulse could be applied to a chlorided silver electrode close to the long
segment. Current flow across the rubber partition was
recorded through a similar electrode close to the short
segment and connected to an operational amplifier in
virtual ground configuration. Stimulation threshold for the
preparation varied between 1 and 5 /AA.
SOLUTIONS AND SOLUTION CHANGES
The control Ringer's solution applied to both segments
had the following (millimolar) composition: sodium chloride, 110; sodium bicarbonate, 2.4; potassium chloride,
2.5; calcium chloride, 0.7; dextrose, 7.5. Fine bubbles of a
99% O2, 1% CO2 mixture were passed through the solution, giving a pH of 7.2. Before voltage clamp procedures,
the long segment was equlibrated with a Ringer's solution
in which 98% of the sodium was isosmotically replaced by
choline as the chloride. The freezing points of both solutions were equal. In the experimental maneuvers described below, the calcium concentration was changed
(0.1-2.0 ITIM) or manganous chloride added (to 1.0 and
10.0 rr>M), as indicated, in the Ringer perfusing the short
segment. The Ringer bicarbonate concentration used in
these experiments was kept extremely low in order to
maximize the attainable concentration of manganous ion
and to achieve large and rapid negative inotropic effects.
Thus the true chemical activity of the calcium ion may be
higher than occurs in the presence of large amounts of
bicarbonate buffer. This might amount to an increase in
true calcium activity of as much as 25%, as compared with
a medium buffered with 24 mM bicarbonate, for example,
according to Schaer.23 Experiments were conducted at
room temperature, which varied between 23°C and 26°C;
but the Ringer temperature did not vary by more than
0.5cC in any one experiment.
The solution perfusing the short segment could be
changed rapidly by turning a stopcock located close to the
jet. Since the force of the jet against the muscle could
474
CIRCULATION RESEARCH
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contribute significantly to its resting tension, it was necessary to ensure that changes in muscle length due to slight
differences in the forcefulness or position of the jet were
not responsible for observed alterations in contraction
after switching solution. Equal hydrostatic heads of pressure and equal flows for all solutions (as judged from
rotometer flowmeters in the lines) helped to minimize
such effects, as did holding the short segment near its
length-tension plateau, where contraction is relatively insensitive to length. In addition, a delay between the stopcock and the tip of the jet of about 2 seconds was maintained, so that the force record could be seen to return to
its original time course after the switching artifact, but
before the new solution reached the muscle.
Maximal rate of rise of the action potential upstroke,
measured close to the rubber partition, was unchanged
after perfusing the long segment with the low sodium
Ringer's solution. It thus seems likely that significant extracellular sodium gradients did not extend into the short
segment. Action potential duration was, however,
shortened to varying degrees, depending on the extracellular leakage conductance, and this was accompanied by a
reduction in time-to-peak and developed force of the associated contraction. Leakage was sometimes very high, but,
most frequently, action potentials measured across the
rubber partition ranged between 40 and 70 mV in amplitude. This was found with fair regularity when the hole in
the partition was made very small, constricting the muscle
from a diameter of 0.9 mm to one of 0.35 mm. Such a
profound constriction of the muscle interfered seriously,
VOL. 41, No. 4, OCTOBER
1977
however, with rapid penetration of its extracellular compartment by the perfusing medium on changing solutions.
For most of the experiments reported here, for which
rapid access of the Ringer medium to the interstitial spaces
was important, the hole in the rubber partition was not as
constricting, being reduced only to 0.65 mm in diameter.
DEGREE OF SPACE CLAMP
The uniformity of voltage distribution achieved during
clamp is of critical importance for the main conclusions of
the present study. This was directly monitored, therefore,
with an additional microelectrode impalement at a point
on the short segment distal to the feedback electrode.
Figure 2 shows one such result, for a two-step clamp: the
first beyond threshold for the fast inward current, but
short of that for contraction; the second to a very high
inside positive potential. Although voltage is poorly controlled at the distal monitoring site with the first clamp
step (to - 4 9 mV inside), it rises very promptly with the
second clamp step, coming close to its final value within
less than 1 msec and reaching that value within 10 msec.
The falling off in voltage from the site of the feedback
electrode to the more distal one of the monitoring electrode amounts to 15%, both for the control Ringer and for
that in the presence of 10 mM manganous ion. In three
additional preparations, the falloff in voltage from the
feedback electrode to a more distal monitoring electrode
was found to be of comparable magnitude, amounting to
7-10% over distances of 0.11-0.14 mm, all of which
comprised 3/io of the total length of the short segment.
VOLTAGE TIME COURSE, DURING CLAMP, AT:
FEEDBACK ELECTRODE
MONITORING ELECTRODE
0.12mm (0.3 « SHORT SEGMENT
LENGTH) FROM PARTITION
0.28mm (0.7 » SHORT SEGMENT
LENGTH) FROM PARTITION
CLAMP PRIOR TO
EXPOSURE TO
lOmM M n " RINGER
SUBSEQUENT CLAMP,
DURING EXPOSURE TO
lOmM M n " RINGER
FIGURE 2 Test of longitudinal uniformity of transmembrane potential during voltage clamp to a markedly inside positive potential. The
entire time course of the two-step clamp is shown, in each of the four sets of traces, on a slow swep (2.4-second calibration); the two
depolarizing steps and the two repolarizing steps are drawn in, in each case, as dashed lines. The onset and early time course of the second step
is shown, in each set, on a faster trace (48-msec calibration) with the upstroke drawn in as a lighter dashed line and at a greater amplifier gain.
When the transmembrane potential at the feedback electrode (traces in left column) is displaced to +115 mV inside, the voltage monitored at a
more distal site (traces in right column) can be seen to be only +85 mV inside. This voltage distribution is the same in the control Ringer
(upper row of traces) and in the presence of JO mM manganous chloride (lower row).
ACTIVATION OF CONTRACTION IN FROG VENTRICLEAWerson et al.
475
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beat to almost zero force (in 0.1 mM Ca), to 94% of its
maximal value (in 2.0 mM Ca) and back to the exact
control level (in 0.7 mM Ca). This occurred rarely. A
result more typical of these experiments and those of
C." 0 . 1 * U"
others12-16 is shown in Figure 3B, where a new steady state
is not quite attained after four beats at 30-second intervals. To a large extent, the beat-by-beat progress to a new
steady state could be retarded or speeded up by adjusting
the manner in which the jet made contact with the short
segment, even after several hours of elapsed experimental
time. Rapidity of change in contraction was promoted, in
0.7rflC ~
2.0 * C."
general, by not making the silk snare or the hole in the
FIGURE 3 Rapidity of extracellular access. A, Record of contrac- rubber partition any tighter than was necessary to hold the
tile force development at a stimulation rate of 12/min. Changes in short segment securely. In addition, a small reduction in
calcium concentration in the Ringer medium occuring between two its length from the optimal one for contraction allowed the
beats are followed by contractions at, or almost at, new steady state
segment to arc very slightly in the Ringer jet and to spread
levels. Arrows indicate stimuli (which elicited action potentials)
apart the fascicles in the spongy structure of the muscle.24
during exposure to 0.1 mu Ca Ringer. The stopcock was turned
Even when such maneuvers were painstakingly carried
just before a beat, so that the new Ringer, according to a carefully
measured delay time, would strike the muscle after that beat. B, A out, muscles varied widely in the promptness of their
more typical case, in which a new contractile level is reached four contractile responses to a change in Ringer calcium. In
beats after a change in calcium concentrationfrom0.7 to 2.0 mu. Figure 4, the fraction of the steady state change achieved
This frog ventricle strip was stimulated at a rate of2/min.
in the first beat in the calcium-rich medium is plotted for
11 preparations. In general, this fraction increases as the
period of exposure to the new Ringer (i.e., the cycle
length) is prolonged. This result is most easily understood
Levels of clamp in these preparations were to +5 mV,
as indicating that the technical problem of access of the
+ 43 mV, and +106 mV, respectively. Finally, the extraaltered Ringer's fluid to the interstitial spaces of the muscellular voltage difference during clamp, for the preparacle
makes a large contribution to the observed rapidity of
tion shown in Figure 2, was small and amounted to 4 mV
the change in contraction. Other possibilities are, howfor the clamp level shown.
ever, not excluded by this finding, such as a rate-limiting
Voltage control achieved during clamps in the region
step of binding (or unbinding) of extracellular calcium to
between the voltage threshold for the fast inward current
some sarcolemmal site. Also, technical limitations on exand that for contraction was quite variable from one preptracellular access may actually obscure an underlying pararation to another. In some it was poor (for example, the
ticipation of intracellular calcium stores in the beat-byfirst clamp step for the preparation shown in Figures 2,13,
beat response to a change in Ringer calcium concentraand 14), while, in others, uniform voltage control was
tion.
rapidly attained and thus no phasic contractions were
visible during the first step (preparations shown in Figures
9, 10, and 15). Contraction during the second clamp was
not influenced by the introduction of a prior depolarization, as was reported previously.20 The use of the two-step
clamp provides, with the second step, a rapidly attained
voltage of fair distribution prior to force development, as
documented in Figure 2, and this procedure was used in all
maneuvers where contractile responses at high inside positive potentials were studied (results shown in Figures 9-11
and 13-15).
Results
RAPIDITY OF THE EFFECT ON CONTRACTION OF
CHANGES IN EXTRACELLULAR CALCIUM
CONCENTRATION
If the activation process in frog ventricle were mediated
exclusively by a transsarcolemmal influx of calcium, one
might expect the contraction to reach a new steady state in
the beat immediately following an increase in the calcium
concentration of the Ringer's solution. This might occur if
one were able to alter, rapidly and uniformly, the extracellular calcium concentration. Such a result is shown in
Figure 3A, where a change in calcium, initiated very early
in the 5-second interval between beats, brings the next
CYCLE LENGTH (SEC)
FIGURE 4 Variability of extracellular access in 11 preparations.
Each point represents the increase in contractile force, in the first
beat after changing from 0.7 mM to 2.0 mM Ca Ringer, as a
fraction of the total increase attained in the steady state. Each line
connects the values obtained from a single muscle preparation for
different time intervals between beats.
476
CIRCULATION RESEARCH
100 He
vj
V.j
0 . 7 rfl C*** RIKGEfi
n
V.J
I
2.0 *
(->•• RINGER
FIGURE 5 Effects on subsequent contractions of an abrupt rise in
intracellular calcium concentration (stimulated twitch, followed by
three voltage clamp-induced contractions) and those of an increase
in extracellular calcium concentration (the last three clamps, in the
Ca-rich medium). Traces (force above, transmembrane potential
below) are interrupted to show compression of the time axis (slower
chart speed) in the period between pulses, which were delivered
every 20 seconds.
VOL. 41, No. 4, OCTOBER
1977
That the effect shown in Figure 6 cannot be attributed to
a calcium conductance, which is maximally activated at an
unusually large inside positive potential, is shown by the
experiment illustrated in Figure 7. Here the plateau is
displaced, by a 100-msec pulse, to a level (+107 mV)
which should be well beyond the equilibrium potential for
calcium and, subsequently, to a level still more positive
( + 140 mV). Whether the pulse is applied during the
upstroke (left traces) or 100 msec after the upstroke (right
traces), the associated contraction is augmented, relative
to the control, and the augmentation is greater, the more
positive the value to which the transmembrane potential is
displaced. The graph in Figure 8 gives all of the results
obtained in an experiment of this type, for displacements
over the range from the naturally occurring plateau to the
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LACK OF A DELAYED INOTROPIC EFFECT DUE TO
AN INCREASE IN EXTRACELLULAR CALCIUM
CONCENTRATION
The problem of access, then, may account totally for the
beat-by-beat response of frog ventricle to a change in
Ringer calcium concentration; or it may, alternative, obscure a significant contribution to the activation process of
intracellular calcium storage and subsequent release. Since
extracellular composition cannot, apparently, be controlled at will with any regularity, this alternative possibility was assessed in an indirect manner. A series of regularly repeating stimulus pulses was suddenly replaced by a
train of 4-second clamp pulses, at the same repetition rate
(Fig. 5). Contraction with the first clamp pulse was more
than 10-fold greater than that accompanying the preceding
action potential, and more than 6 times as long-lasting. It
is thus reasonable to infer that there was a large increase in
free intracellular calcium concentration which did not lead
to any further increase in contraction with two subsequent
clamp pulses, after which an increase in extracellular calcium concentration was followed by the characteristic,
beat-by-beat, progressive increase in contraction during
three clamp pulses.
100
FIGURE 6 Inotropic effects of displacement of transmembrane
potential (lower traces) during the action potential plateau. Current
pulses making the cell interior more positive (1) and more negative
(2) by about 5 mV were applied 2-3 msec after completion of the
upstroke. Contractile force traces (upper traces, increased tension
is in downward direction) are numbered correspondingly. The control, unmodified action potential and contraction, omitted for clarity, had time courses intermediate to I and 2.
100msec PULSE, STARTING:
ACTIVATION OF CONTRACTION DURING THE
ACTION POTENTIAL PLATEAU
A polarizing pulse, applied after completion of the action potential upstroke, displaced the plateau to a more
inside positive or inside negative value for a period of
about 300 msec (Fig. 6). These maneuvers would correspond to a decrease (traces 1) or an increase (traces 2),
respectively, in the driving force for a calcium current. The
accompanying contractions were, however, oppositely affected, being increased when the plateau was made more
inside positive and decreased by the reverse maneuver.
The effect could be graded by varying the duration of the
polarizing pulse from 30 msec to that of the entire plateau.
These results are in marked contrast to those obtained by
Sumbera25 for trabeculae from sheep and calf ventricles
and by Vassort and Rougier26 for frog atrial trabeculae.
Both found contraction to be changed in the same direction as the electrodiffusional driving force on calcium and
with the effect confined to the early time course of the
action potential.
100msec AFTER
UPSTROKE
DURING
UPSTROKE
+ l40mV
+ IO7mV
+30mV
30mg
I sec
FIGURE 7 Inotropic effects of extreme displacement of the action
potential plateau by 100-msec polarizing pulses. Superimposed
traces of the unmodified (+ 30 mV) and modified (to +107 and
+ 140 mV) action potentials are shown for pulses starting during
the upstroke (left) and 100 msec after the upstroke (right). The
associated contractions are shown in the superimposed traces below, where peak tension can be seen to increase with increasing
inside positive levels of the displaced action potential plateau.
ACTIVATION OF CONTRACTION IN FROG VENTRICLE/Anderson et al.
@2OOm
+3OmV
+60mV
+90mV
+!20mV
TRANSMEMBRANE POTENTIAL ATTAINED
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FIGURE 8 Relationship between the degree of displacement of the
action potential plateau by a 100-msec pulse and the augmentation
of the accompanying contraction. The overshoot of the unmodified
action potential amounted to +25 mV. Each plotted symbol gives
the peak force attained, for a given displaced level of the action
potential plateau (abscissa) as a multiple (ordinate) of that attained
in control beats bracketing the experimental maneuver. Solid lines
connect the mean values for each group of points, which represent
results for application of the polarizing pulse during (closed circles), 100 msec after (crosses), and 200 msec after (open circles),
the upstroke of the action potential.
169 n>
•It
MV
204 to
•33 nv
213 As
221 fls
• 5 0 MV
•64 MV
225 to
•72 nv
219 to
•83 (iv
213 to
•98 HV
-46 nv
-83 nv
FIGURE 9 Contractile response (upper trace) to increasing depolarizations (lower trace) in 0.7 mM Ca Ringer. Interrupted portions
of the traces show a slowing of chart speed during the period
between two-step clamp pulses, which were applied every 20 seconds. Total duration of each clamp is indicated. The second clamp
step followed the first by 1 sec and was 1.6 seconds in duration.
large inside positive levels shown. Each experimental
point was bracketed by control contractions during which
no polarizing current was passed during the plateau. Without exception, displacement of the plateau toward a more
inside positive value enhances contraction, and the degree
of augmentation is monotonically related to the extent of
voltage displacement.
477
levels between +33 and +50 mV (Fig. 9), while, as
reported previously,20 contraction in 0.2 mM Ca Ringer
increases monotonically over a considerably greater range
of inside positive potentials (Fig. 10). In addition, the
voltage-tension relationship can be changed from one type
to the other by altering the extracellular calcium concentration (Fig. 11).
In Figure 9 it can also be seen that, while a transmembrane potential of —46 mV is the approximate mechanical
threshold before the second clamp step, it is no longer so
after the second step and its accompanying large contraction. This effect was seen frequently at clamp levels which
produced saturation, or near saturation, of force development.
NATURE OF ACTIVATION AT LARGE INSIDE
POSITIVE POTENTIALS
Whether force development induced by extreme levels
of intracellular electropositivity is also strongly and rapidly
dependent on extracellular composition is of special interest, since such voltage levels appear to lie beyond the
equilibrium potential for calcium and thus bring about a
net outward electrodiffusional driving force on this ion.
That the entire extent of the intracellular compartment
had been clamped to such voltage levels was supported, in
a few instances, by an additional impalement to monitor
voltage distribution (see Figure 2 and related text: Methods, Degree of Space Clamp). That contracile force developed at such highly positive intracellular potentials is mediated by calcium influx into the cells was tested by abrupt
changes in Ringer composition which are known to affect
calcium influx, i.e., addition of manganous ion,27 increase
in calcium concentration, and decrease in sodium concen-
0.7mM Co"
o
IE
o
Q
LJ
O
-40
+40
+120
TRANSMEMBRANE POTENTIAL (mv)
SATURATION OF THE ACTIVATION PROCESS
In 0.7 mM Ca Ringer, long clamp pulses elicit a maximum or near maximum of force development at voltage
FIGURE 11 Voltage-tension relationship at two extracellular calcium concentrations, in the same preparation. Data obtained as in
Figures 9 and 10.
FIGURE 10 Contractile response to increasing depolarizations in 0.2 mM Ca Ringer. Transmembrane potential time course is above and
associated contraction below. As in Figure 9, chart speed is slowed between two-step clamps which were applied at 20-second intervals. In
each case the second step occurred 0.6 second after the first and was 2.6 seconds in duration.
478
CIRCULATION RESEARCH
No. 1 : NORMAL RINGER
T
100
MV
I
No. 2: 50Z H. RINGER
Nn. V. NO8MAI BIHKFR
No. 1: NORMAL RINGER
FIGURE 12 Rapid effects, on contraction due to manganous ion
(upper traces) and to a decrease in extracellular sodium concentration (lower traces). Contractions were induced by voltage clamp
pulses (middle trace) delivered at a frequency of3lmin. Numbers
indicate consecutive contractile responses, the indicated change in
Ringer composition having occurred just after the preceding clamp
pulse.
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0
•,
0.2rttC o RINGER
10 SEC
-49
0.2 tf
0.2 Mfl Co -, '10 Nfl fin*
1NGER
SINGER
A.
FIGURE 13 Rapid and promptly reversible effect of extracellular
manganous ion on force (lower trace) developed at a high inside
positive potential (upper trace). T, a test of resting potential recording on switching off feedback; ad], repositioning of baseline; C,
reimposition of clamp feedback. Chart speed is constant. The first
two clamps are the same ones shown in Figure 2, where voltage
monitored at a more distal point is also displayed. Clamp durations
are as in Figure 2: total duration of each clamp pulse was 3.8
seconds; interval between onset of first and second clamp steps was
1.0 seconds; duration of second step was 2.4 seconds.
tration.28 The drugs verapamil and D-600 were also tried,
but these required several beats to have appreciable effects which were very slowly reversed, on withdrawal of
the drug. Changes in sodium concentration and addition of
manganous ion, in contrast, had the extremely rapid effects on contraction shown earlier for alterations in calcium level (Fig. 12).
0.58
VOL. 41, No. 4, OCTOBER
1977
Voltage distribution during clamp was found to be quite
variable, especially at large inside positive potentials. It
was, therefore, thought advisable to verify longitudinal
uniformity of voltage in the same preparation and during
the same clamp pulses, from which the most critical observations of contraction were obtained. Thus, the two-step
clamps whose voltage uniformity is given in Figure 2 are
the first two clamps in Figure 13, which shows that application of 10 HIM Mn2+ Ringer immediately after the first
clamp reduced the developed force of the next contraction
almost to its steady state value. Withdrawal of the 10 mM
Mn2+ was followed by a complete return to the control
value with the next contraction. Rate of force development was even more profoundly decreased by the manganous ion than was peak force. In five other preparations, in which voltage distribution was not monitored,
more dramatic decreases in contraction were observed.
Similarly, the force developed at a large inside positive
potential was also rapidly sensitive to a change in extracellular calcium concentration (Fig. 14). Switching to 50%
Na Ringer between two clamps at a high level of electropositivity does not affect the peak level of force development (Fig. 15). This is as would be expected from the information given above that saturation of the activation
process is present at this voltage level in 0.7 mM Ca2+. The
rate of force development, however, was markedly
speeded up, bringing contractile force to its peak in about
half the control time.
10 SEC
2.0 «M
0 . 2 MM Co*
RINGER
CORINGER
FIGURE 14 Effect of an increase in extracellular calcium concentration of force developed at a high inside positive potential. Same
preparation, same display, and same clamp durations as in Figure
13.
8/SEC
0.91 8/SEC
338
0.7 C++
332 ng
RINGER
0.7 C++ , 501 No++
RINGER
103 MV
FIGURE 15 Effect of a decrease in extracellular sodium concentration on force (upper traces, increased tension is in downward direction)
developed at a high inside positive potential. Two consecutive clamps are shown, which precede (left) and immediately follow (right) a change
in Ringer composition, at a frequency ofUmin. Total duration of each clamp pulse was 3.8 seconds; interval between onsets offirstand
second clamp steps was 0.7 seconds; duration of second step was 2.2 seconds.
ACTIVATION OF CONTRACTION IN FROG VENTRICLE/Anderson et al.
Discussion
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These results strongly suggest that activation of contraction in frog ventricular muscle is mediated by an influx of
calcium into the cell. They do not totally exclude the
participation of intracellular calcium binding sites, but
they do indicate that the failure to attain a new steady state
in the beat immediately following alteration of extracellular concentration is not attributable to beat-by-beat increases in binding and release of calcium at such sites. An
immediate new steady state of contraction was attained in
a minority of preparations (Fig. 3A). In the great majority, where this did not occur, an abrupt and large increase
in intracellular free calcium concentration, caused by a
prolonged voltage clamp pulse, had no residual effect on
subsequent contractions. It is, therefore, reasonable to
explain the progressive beat-by-beat increase in contraction as being largely due to limitations of technique in
terms of the rapidity and uniformity with which extracellular composition can be changed in most preparations.
In addition, the abrupt abolition of tension, during a
prolonged contraction, by sudden reduction in Ringer calcium concentration21 argues against a significant role for
calcium-induced release of calcium from intracellular storage sites,29' 30 since the contraction is more sensitive to the
altered extracellular calcium level than it is to the high
concentration of intracellular free calcium reflected by the
large developed force.
This finding does not exclude the participation of calcium release from such sites earlier on in the development
of activation. In addition, the cyclic behavior of contraction which can be a consequence of regenerative release of
calcium from storage sites was not observed by Fabiato
and Fabiato31 in preparations of frog ventricle from which
the sarcoiemma had been removed, in contrast to mammalian preparations from several species. Spontaneous
contractions of much slower time course and frequency
were observed by Winegrad32 in a frog ventricle preparation from which the sarcoiemma was not removed but
rendered highly calcium-permeable by exposure to
EDTA.33
It does not seem likely that all of the reported observations on the beat-by-beat time course of contraction of
frog ventricle can be dismissed as arising from a slowness
of extracellular access. The contractile response to
changes in Ringer calcium is extremely slow for a considerable period following a prior exposure to a calcium-poor
medium, according to Chapman and Niedergerke,16 and
this was consistently observed in the present experiments.
In addition, staircase and the frequency-force relationship
have been observed countless times in this tissue. In view
of the findings presented here, however, it may be as
reasonable to attribute such effects to progressive alterations in transsarcolemmal calcium flux as to the kinetics of
calcium binding to, and release from, intracellular sites.
An explanation of this sort would also be consistent with
the very great and almost instant sensitivity of contraction
to alterations in extracellular calcium concentration.21
Also, the results presented here do not argue against an
important effect on activation of mechanisms which might
remove calcium from its site of action, either through
intracellular binding33 or extrusion from the cell.34
479
The calcium influx that mediates activation does not
have the characteristics of a calcium current. If, in this
tissue, a calcium current does exist (cf. Morad and Orkland20), it does not appear to be a major determinant of
contractile activation. This can be concluded from the fact
that displacements of the action potential plateau, which
should alter (and even reverse) the driving force for such a
current, affect contraction in a manner opposite to that to
be expected, even when they are confined to an early
portion of the plateau. The early and later portions of the
contractile time course do not differ from ,one another in
terms of the sensitivity of contraction either to displacements of transmembrane potential (Figure 8 and related
text) or to changes in extracellular calcium concentration.21 Thus, if more than one mechanism mediates excitation-concentration coupling in mammalian ventricle8-25
and in frog atrium,26- 35- 36 there is as yet no evidence that
this is the case in frog ventricle .20
For the contraction accompanying a conventionally
stimulated action potential, activation is close to being
saturated in 2.0 ITIM calcium Ringer. The calcium concentration of 0.7 mM was chosen for the control medium since
this gives a contractile force close to the foot (20-30% of
the way to peak) of the sigmoid calcium-force curve.
During prolonged clamp pulses, which elicit a plateau of
contractile force, near saturation of activation occurs at
+ 33-+50 mV in 0.7 mM calcium Ringer and at +120 mV
in 0.2 mM calcium Ringer. Saturation or near saturation
was consistently observed in several experiments at each
calcium level. The reason for the contrary report of Morad
and Orkand20 is not clear. It should be noted that space
clamp is relatively nonuniform in frog ventricle, and we
were at some pains to be sure that poor voltage distribution did not invalidate the observations made at high
inside positive potentials. Conceivably, in preparations
longer than those used here, a greater voltage difference
along the muscle might obscure a saturation effect.
Although the possibility has not been excluded that it is
sarcolemmal calcium transport which is being saturated, it
is reasonable to assume that this effect occurs at the level
of activation. Accordingly, if one takes the 0.35-mm hole
size (in the rubber partition) as the diameter of compact
frog ventricular muscle, the average developed force, for
10 preparations at high inside positive potentials amounts
to 360 mg. This gives a value of 4 g/mm2, compared to a
"contractile maximum" of about 6 g/mm2 for cat ventricular trabeculae.37
Thus, at +120 mV, myoplasmic calcium concentration
should be close to 10"5 M, certainly in excess of 10"6 M.33
For the latter concentration, the calcium reversal potential
in 0.2 mM calcium Ringer amounts to +69 mV. As shown
in Figure 2, the transmembrane potential is displaced from
below the threshold for contraction to beyond the calcium
reversal potential within a few milliseconds. Force development, which occurs following the completion of this
voltage displacement (Fig. 13, first two clamps), is extremely sensitive to the application of manganous ion to
the muscle, which was only begun in the 16-second period
of "diastole" prior to that depolarization. It thus appears
unlikely that manganous ion is exerting its negative inotropic effect at an intracellular site. A more plausible
480
CIRCULATION RESEARCH
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interpretation of this result is that manganous ion is blocking calcium influx into the fibers in a manner analogous to
that well demonstrated for calcium currents.27 Activation
of contraction at such high inside positive potentials,
therefore, appears also to be mediated, at least in part, by
a transmembrane calcium flux, whose driving force includes components in addition to an electrodiffusional
one. The nature of such an additional component of the
driving force is not clear. One possibility is that of counterflow, the linking of calcium influx to efflux of some other
ion through affinity for a common membrane carrier, as
formulated for uncharged molecules by Rosenberg and
Wilbrandt.38 Sodium efflux may play such a role at such
extremely inside positive potentials, and the result shown
in Figure 15 is in accord with this possibility, but this result
might, alternatively, be attributed to a decrease in the rate
of calcium extrusion from the fibers. The possibility that
calcium influx may be linked to potassium efflux,22 which
would provide such an additional driving force for all
degrees of depolarization from the potassium equilibrium
potential, remains as yet to be evaluated. In its simplest
form, at least, it faces two difficulties: first, elevation of
extracellular potassium concentration has a positive, voltage-independent inotropic effect;39 and, second, the addition of potassium chloride to Ringer's solution without
reduction of sodium concentration causes large contractures,24 although little or no outward driving force on
potassium has been created.
References
1. Marey M: Physiologie Experimental, vol. 2, Paris, Masso, 1876
2. Antoni H, Jakob R, Kaufmann R: Mechanische Reaetionen des
Frosch und Saugetiermyokards bei Veranderung der Aktionspontential Dauer Durch Konstant Gleichstromimpulse. Pfluegers Arch 306:
33-57,1969
3. Edmands RE, Greenspan K, Bailey JC: Role of the premature action
potential in contractile potentiation; a study of paired stimulation.
Cardiovasc Res 6: 368-374, 1972
4. Kavaler F, Anderson TW: Delayed inotropic effects of membrane
depolarization in ventricular muscle. In Myocardial Biology, vol. 4,
edited by NS Dhalla. Baltimore, University Park Press, 1974, pp 2129
5. Woodworth RS: Maximal contraction, "staircase" contraction, refractory period, and compensatory pause of the heart. Am J Physiol 8:
213-249, 1902
6. Hoffman BF, Bindler E, Suckling EE: Postextrasystolic potentiation
of contraction in cardiac muscle. Am J Physiol 185: 95-102, 1956
7. Braveny P. Kruta V: Dissociation de deux facteurs: Restitution et
potentiation dans Faction de rintervalle sur I'amplitude de la contraction du myocarde. Arch Int Physiol 66: 633-652, 1958
8. Beeler GW Jr, Reuter H: The relation between membrane potential,
membrane currents and activation of contraction in ventricular myocardial fibres. J Physiol (Lond) 207: 211-229, 1970
9. Wood EH, Heppner RL, Weidmann S: Inotropic effects of electric
currents. Circ Res 24: 409-445, 1969
10. Ochi R. Trautwein W: The dependence of cardiac contraction on
depolarization and slow inward current. Pfluegers Arch 323: 187-203.
1971
11. Sands SD, Winegrad S: Treppe and total calcium content of the frog
VOL. 41, No. 4, OCTOBER
1977
ventricle. Am J Physiol 218: 908-910, 1970
12. Page S, Niedergerke R: Structures of physiological interest in the frog
heart ventricle. J Cell Sci 11: 179-203, 1972
13. Sommer JR, Johnson EA: Cardiac muscle: a comparative ultrastructural study with special reference to frog and chicken hearts. Z Zellforsch Mikrosk Anat 98: 437-468, 1969
14. Staley NA, Benson ES: The ultrastructure of frog ventricular cardiac
muscle and its relationship to mechanism of excitation-contraction
coupling. J Cell Biol 38: 99-114, 1968
15. Niedergerke R: The staircase phenomenon and the action of calcium
on the heart. J Physiol (Lond) 134: 569-583, 1956
16. Chapman RA, Niedergerke R: Effects of calcium on the contraction of
the hypodynamic frog heart. J Physiol (Lond) 211: 389-421, 1970
17. Chapman RA, Miller DJ: The effects of caffeine on the contraction of
the frog heart. J Physiol '(Lend) 242: 589-613, 1974
18. Weidmann, S: Effect of increasing calcium concentration during a
single heartbeat. Experientia IS: 128-129, 1959
19. Kavaler F, Fisher VJ, Stuckey JH: The potentiated contraction and
ventricular "contractility." Bull NY Acad Med 41: 592-601, 1965
20. Morad M, Orkand RK: Excitation-contraction coupling in frog ventricle: evidence from voltage clamp studies. J Physiol (Lond) 219: 167189, 1971
21. Kavaler F: Electromechanical time course in frog ventricle: manipulation of extracellular calcium concentration during voltage clamp. J Mol
CeU Cardiol 6: 575-580, 1974
22. Kavaler F, Morad M: Paradoxical effects of epinephrine on excitationcontraction coupling in cardiac muscle. Circ Res 18:492-501,1966
23. Schaer H: Influence of respiratory and metabolic acidosis on epinephrine-inotropic effect in isolated guinea pig atria. Pfluegers Arch 347:
297-307, 1974
24. Lamb JF, McGuigan JAS: Contractures in superfused frog's ventricle.
J Physiol (Lond) 186: 261-283, 1966
25. Sumbera J: Induced changes of action potential on cardiac contraction. Experientia 26: 738-739, 1970
26. Vassort G, Rougier O: Membrane potential and slow inward current
dependence of frog cardiac mechanical activity. Pfluegers Arch 331:
191-203, 1972
27. 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
28. Niedergerke R: Movements of Ca in frog heart ventricles at rest and
during contractures. J Physiol (Lond) 167: 515-550, 1963
29. Endo M, Tanaka M, Ogawa Y: Calcium induced release of calcium
from the sarcoplasmic reticulum of skinned skeletal muscle fibres.
Nature 228: 34-36, 1970
30. Ford LE, Podolsky RJ: Regenerative calcium release within muscle
cells. Science 167: 58-59, 1970
31. Fabiato A, Fabiato F: Activation of skinned cardiac cells. Eur J
Cardiol 1/2: 143-155, 1973
32. Winegrad S: Spontaneous mechanical activity in depolarized frog
ventricle. J Gen Physiol 68: 145-157, 1976
33. Winegrad S: Studies of cardiac muscle with a high permeability to
calcium produced by treatment with EDTA. J Gen Physiol 58: 71-93.
1971
34. Benninger C, Einwachter HM, Haas HG, Kern R: Calcium-sodium
antagonism on the frog's heart: a voltage-clamp study. J Physiol
(Lond) 259: 617-645, 1976
35. Einwachter HM, Haas HG, Kern R: Membrane current and contraction in frog atrial fibers. J Physiol (Lond) 227: 141-171, 1972
36. Leoty C, Raymond G: Mechanical activity and ionic currents in frog
atrial trabeculae. Pfluegers Arch 334: 114-128, 1972
37. Kavaler F, Marlon A, Harris RS. Fisher VJ: "Contractility" in the
excised and the in situ papillary muscle. In Paired Pulse Stimulation of
the Heart, edited by PF Cranefield, BF Hoffman. New York, Rockefeller University Press, 1968, pp 72-81
38. Rosenberg T, Wilbrandt W: Uphill transport induced by counterflow.
J Gen Physiol 41: 289-296. 1957
39. Kavaler F. Hyman PM. Lefkowitz RB: Positive and negative inotropic
effects of elevated extracellular potassium level on mammalian ventricular muscle. J Gen Physiol 60: 351-365. 1972
Mechanism of activation of contraction in frog ventricular muscle.
T W Anderson, C Hirsch and F Kavaler
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Circ Res. 1977;41:472-480
doi: 10.1161/01.RES.41.4.472
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