Download Original Contributions Comparison of the Effects of Different

1161
Original Contributions
Comparison of the Effects of Different
Inotropic Interventions on Force, Velocity,
and Power in Rabbit Myocardium
Y. Christopher Chiu, Keith R. Walley, and Lincoln E. Ford
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To gain some insight into inotropic mechanisms, we compared the effects of several classes of
inotropic interventions on the isometric twitch and force-velocity properties of isolated rabbit
myocardium. Postextrasystolic potentiation was chosen as one of the interventions in the belief
that its onset is so rapid that it would be unlikely to cause substantial chemical changes in the
contractile proteins and that most of the effects would be due to changes in the level of
activation. The effects of a digitalis analogue (acetylstrophanthidin), an adrenergic agent
(isoproterenol), and a methylxanthine (caffeine) were then compared with those of postextrasystolic potentiation. The conditions were chosen so that each agent caused a twofold increase
in twitch force. Acetylstrophanthidin and postextrasystolic potentiation caused twitch force to
increase with only a slight (11%) decrease in time to peak force. Isoproterenol caused the peak
of the twitch to occur substantially (40%) earlier with marked abbreviation of the twitch.
Caffeine had the opposite effect: time to peak force was delayed (by 60%), and the twitch was
markedly prolonged. In contrast to the marked differences in the time course of the twitch,
there was no significant difference between the instantaneous force-velocity curves obtained with
the different interventions. All four interventions caused maximum velocity to increase slightly
(1-9%) and maximum power to increase only slightly more than twitch force (5-21%). All of
the changes observed can be accounted for by changes in activation, either by an increase in the
amount of calcium released into the myonlament space or by a change in the sensitivity of the
myonlaments to calcium. There was no need to postulate direct changes in the contractile
machinery to account for these results. (Circulation Research 1989;65:1161-1171)
I
t is well known that different inotropic interventions have different effects on the shape of the
twitch contractions in isolated cardiac muscle.1
This raises the question of whether the different
interventions alter the extent of muscle activation
or whether they also change the contractile response
of the activated myonlaments. For technical reasons, previous efforts to address this question have
not given unimpeachable results. The studies were
done with mechanical levers that produced sustained oscillations when the load was suddenly
changed. To avoid these oscillations, the earliest
studies used "afterloaded contractions" to obtain
individual points on a force-velocity curve. 2 - 4 The
From the Cardiology Section, Department of Medicine, the
University of Chicago, Chicago, Illinois.
Supported in part by US Public Health Service Grant HL29298, by a fellowship from the Chicago Heart Association
(Y.C.C.), and by a fellowship from the Frances B. Parker
Foundation (K.R.W.).
Address for correspondence: Lincoln E. Ford, MD, Box 249,
The University of Chicago Hospitals, 5841 South Maryland
Avenue, Chicago, IL 60637.
Received December 7, 1988; accepted May 9, 1989.
ends of the muscles were held at a fixed length
during the onset of a twitch until the force in the
muscle rose to the value of an afterload, determined
by a weight hung from a lever attached to the
muscle. After that time, the muscle shortened at a
constant load determined by the weight. Either the
initial or the maximum velocity was plotted as a
function of load, and multiple points were obtained
by using different afterloads in different contractions. These experiments were criticized because
each point on the force-velocity curve was obtained
at a different time in the twitch and at a different
sarcomere length.5-7 It was generally recognized
that the correct way to do the experiments was to
release the muscle to different loads at the same
time in different but identical contractions.5 In this
way, the level of activation and the amount of
sarcomere shortening, caused by extension of the
series elastic element as force developed, would
always be the same at the onset of shortening. Until
recently, attempts to make these types of experiments were not technically feasible. Large, poorly
damped oscillation prevented measurement of short-
1162
Circulation Research Vol 65, No 5, November 1989
ening velocity for many tens of milliseconds after
the release,8 and there was some evidence that
these oscillations inactivated the muscle,9-10 although
there is the additional suggestion that this inactivation is caused by the rapid shortening associated
with the quick release and not with the oscillations
themselves. 11 In one study of the effects of
methylxanthines,12 the rapid lever movement was
damped to reduce or eliminate oscillations, but the
force-velocity measurements were not made until a
substantial amount of shortening had occurred after
a steady force was achieved.
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To reexamine the question of the effect of inotropic agents on contractile capability, we have measured the force-velocity properties of rabbit papillary muscle with a servo system capable of
completing critically damped force steps within 1-3
msec.13 This high speed was necessary to make
measurements a few milliseconds after a release
and, thereby, to minimize the cumulative effects of
progressive shortening.11 Our previous experiments
have shown that, even with these rapid steps,
damped recoil of the series elastic elements and
velocity transients preclude measurements earlier
than 5-6 msec after the onset of the step and that
velocity begins to decline almost immediately with
the onset of shortening.11 Thus, there is only a brief
period after a quick release when muscle shortening
velocity can be used to assess the contractile capability at the time of the release. In the present study,
we have measured velocity during a 6—10 msec
period after the onset of the step.
In this study, we have compared the influence of
several inotropic interventions on the isometric
twitch contraction and on the instantaneous forcevelocity relations of rabbit papillary muscle. The
interventions chosen include isoproterenol, which
shortens twitch duration, caffeine, which lengthens
twitch duration, a digitalis analogue (acetylstrophanthidin), which potentiates the twitch without greatly
affecting its time course, and postextrasystolic potentiation. This last intervention was chosen because
its effects are so rapid, within one beat, that there is
little time for a metabolic or biochemical change to
occur and its effects are believed to be due almost
entirely to changes in calcium release.14 The influence of the other interventions on the force-velocity
curves could thus be compared with those of postextrasystolic potentiation to determine whether their
effects could be distinguished from simple increases
in activation.
Materials and Methods
Preparation
Young adult New Zealand White rabbits weighing 2.0-2.5 kg were anesthetized with 100 mg kctamine and 40 mg xylazine, and their hearts were
removed. Right ventricular papillary muscles or
trabcculae having diameters less than 1 mm were
dissected free and mounted in platinum foil clips.13
Hooks on the motor and force transducer were
passed through holes in the clips to attach the
muscles to the apparatus. Throughout the initial
dissection procedure, the hearts and isolated muscles were kept at about 37° C. Once attached to the
apparatus, the muscles were gradually cooled to
room temperature (23°-25° C). In our hands, the
technique of keeping the muscle warm during dissection and mounting13 was much more successful
in yielding muscles that produced good force than
the technique of cooling the hearts rapidly and
performing the dissections near 0° C.16 Muscles that
were dissected at room temperature or in the cold
frequently shortened substantially during dissection
and did not develop much tension.
At the end of the experiment, the muscles were
removed from the apparatus, cut free of the clips,
blotted dry, and weighed. Cross-sectional areas
were determined as the weight divided by 1.05
times the length. The coefficient 1.05 g/cm3 was
taken to be the density of the muscle.
Solutions
All experiments were done in a physiological salt
solution containing (mM) NaCl 118, KC15, NaH2PO4
1.2, NaHCO3 22.5, MgSO4 2, CaCl2 1, and HEPES
5, adjusted to pH 7.4 with NaOH after being equilibrated with 95% O2-5% CO2. During the experiments the solutions were kept in a reservoir where
they were continuously perfused with the same gas.
The solution was fed by gravity through a closed
system of tubing and through the muscle chamber,
which was also closed.
Inotropic agents were added to the solution either
as dry powder (caffeine and acetylstrophanthidin)
or as a x50 concentrated solution (rf/-isoproterenol);
and pH was readjusted after equilibration with the
gas. The final concentrations of these agents were
10 mM caffeine, 11 JAM acetylstrophanthidin, and
4.5 fiM isoproterenol.
Apparatus
The apparatus that has been described previously13 was modified only slightly. Briefly, a servocontrolled linear motor was used to adjust muscle
length. The motor was held horizontally, and a
horizontal length of stainless steel tubing was projected through a seal at one end of the horizontal
muscle chamber. The chamber was 2 mm deep x2
mm wide x 15 mm from the closed end of the trough
where solution entered to the open end of the
chamber, about 3 mm beyond the downstream end
of the muscle. The chamber was covered by a glass
plate, and fluid that was infused into the motor end
was drawn away by suction at the other, open end.
There was a vertical meniscus at the open, downstream end, and a hook from a 2-3 kHz photoelectric transducer17 projected through the vertical meniscus to the clip on one end of the muscle. The
solution flowed through the trough at the rate of
about 1 ml/min. The trough had a cross-sectional
Chiu et al
Comparison of Inotropic Effects in Rabbit Myocardium
area of 4 mm2 so that solution passed the muscle at
a rate of about 4 mm/sec.
The motor could be controlled with a signal from
its own internal position sensor (length control) or
with a signal from the force transducer (force control). Switching between the two types of control
was accomplished by a diode switching network.13-18
Digitized force and length records were obtained
and analyzed with an IBM PC computer equipped
with a Tecmar Labmaster board. The data collection and the timing of the experimental events were
accomplished through this board with the SALT
software package.19-20
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Protocol
Muscles were stimulated every 1.2-2 seconds, a
stimulus interval slightly longer than that required
to produce maximum isometric twitch force. In our
early experiments (most of those with isoproterenol
and acetylstrophanthidin), isotonic steps were
applied every sixteenth twitch; the intervening 15
twitches were used to let the muscle return to a
steady state, and the time was used to store the data
records on disk. With more sophisticated programming, the time between isotonic steps could be
diminished, and every eighth twitch was studied
with an isotonic contraction. When postextrasystolic potentiation was used, it was applied in alternate test contractions, that is, every thirty-second
or sixteenth contraction. In this way, potentiated
and nonpotentiated contractions were studied
together.
After the preparation was mounted, a period of
stabilization was allowed before data were collected. Once the muscles were stabilized, the lengthtension relations were measured to define L ^ , the
longest length at which the developed force-length
relation had a positive slope. The muscle length was
then set at 90% of this value, and all further
measurements were made at this length. This length
was chosen because rest tension was very small
(typically 4-5% of developed force in a control
twitch) and developed force was usually about half
the value at L ^ . Once the length was set, digital
recordings of the isometric twitch were made in
control and postextrasystolic potentiated beats.
From these records, the times to peak force were
determined for both control and potentiated beats.
A set of control and potentiated force-velocity
curves was then made at these times. The reason
for making all the measurements at the time of peak
tension is that our previous studies14 have shown
that the instantaneous contractile parameters (isometric force, maximum velocity, and maximum
power) reach their peak and change least rapidly at
this time. In general, 16 steps to different relative
isotonic loads were applied in 16 different contractions, each separated by seven or 15 isometric
twitches. The effects of postextrasystolic potentiation were evaluated by potentiating every other test
contraction.
1163
The servo system was designed to sense the force
level immediately before the release and to step to a
specified fraction of this value. The fractional step
size, that is, the ratio of isotonic to isometric force,
was increased automatically by approximately 6%
of isometric force, beginning with the lowest load
and progressing to the highest. When the effects of
postextrasystolic potentiation were studied, the step
size was incremented every other step, so that
velocities at the same relative loads were measured
alternately in control and potentiated contractions.
After the data for these force-velocity curves were
obtained, the muscle chamber was perfused with a
solution containing one of the inotropic agents. In
general, a new steady level was reached within 5-15
minutes with all three of the agents. When the new
steady level was achieved, an isometric twitch was
recorded to determine the time to peak tension. The
force-velocity relation at the time of peak isometric
force was measured after the optimum settings for
the servo loop were established. Because of the time
taken to determine the optimum parameters for the
servo loop, as well as the time required for the
muscle to stabilize, the force-velocity measurements
were not made until 20-30 minutes after the solution
was changed. The effects of postextrasystolic potentiation were not measured in the presence of these
agents because it had no additional potentiating
effect in the presence of isoproterenol and caused a
slight decrease in the presence of caffeine. Although
it did further potentiate the twitches in the presence
of acetylstrophanthidin, its effects are not described
here. Similarly, the control experiments were not
repeated after washout of the potentiators because,
with isoproterenol and caffeine, the effects were not
reversible. With isoproterenol, the potentiated force
did not decline substantially over periods of 30-60
minutes after washout. With caffeine, the unpotentiated twitch force fell to baseline levels within a few
minutes of washout, but the twitches were not potentiated by extrasystoles. Both normal and potentiated
twitch force fell to baseline levels within 20-40
minutes after the washout of acetylstrophanthidin,
but to keep the method of analysis consistent, data
from these washout experiments are not included.
Data Analysis
The computer was programmed to collect data at
two different rates. The whole twitch was recorded
at intervals of 5 msec per conversion of both the
length and tension signals. At the time of peak
tension, the interval was decreased to 200 ^usec per
conversion of each signal for 50 msec and then
increased to the slower 5-msec interval to complete
the recording of the twitch contraction (Figure 1).
The slow recording was used to determine the
time-tension integral during the rise of force and the
maximum rate of rise of force. The isotonic force
was determined from the fast records by averaging
the force record over a 4-msec period from 6 to 10
msec after the onset of the step (between dotted
1164
Circulation Research
Vol 65, No 5, November 1989
Velocity=c/(Force+a) - b
where a and b are the force and velocity asymptotes, respectively, of a hyperbola and c is a constant. The parameters of the hyperbola (a, b, and c)
were then converted to the more physiological
parameters Po (isometric force), V ^ , (maximum
velocity), and PV^, (maximum power) by use of
the equations:
Po=c/b-a
V mM =c/a-b
PV m «=ab-l-c-2Vabc
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FIGURE 1. Force and length recordings showing the
time course of the whole twitch at a slow recording speed
and the time course of isotonic shortening at a 25 times
greater recording speed. Ln^, longest length at which the
developed force-length relation has a positive slope. The
transitions between fast and slow recording speeds are
indicated by the interrupted vertical lines. The times over
which force and velocity measurements were made (6-10
msec after the isotonic step) are indicated by the vertical
dotted lines. Muscle length at Lmax was 3.2 mm; crosssectional area was 0.51 mm2.
lines in Figure 1). The velocity was determined by
fitting a linear least-squares regression to the length
record over the same 4-msec period.
Force-Velocity Parameters
The force-velocity data obtained in this manner
were fitted by a nonlinear least-squares (NewtonGauss) procedure to the Hill21 equation:
Typical force-velocity and force-power curves
are shown in Figure 2. The power values were
obtained by multiplying force times velocity.
Slightly different values for the three parameters
could be obtained if the fits were done on the
force-velocity curves or the force-power curves, as
illustrated in Figure 2. When the fits were made to
the force-velocity curves, as in Figure 2A, the
curves passed very close to the low-load highvelocity points. These curves usually passed slightly
below the intermediate points and slightly above the
low-velocity points. When the fits were done in this
manner, the extrapolated isometric force was usually 10—20% greater than peak twitch force, and the
peak of the power curve was slightly less than the
highest measured points (Figure ID). This result
probably occurred because the higher velocities had
larger residuals (larger differences between the data
points and the dotted lines) and the fitting procedure
gave greater weight to these residuals.
When the fits were made to the force-power
curve, as in Figure 2E, a slightly different result was
obtained. The fitted curve lay very close to the peak
B
FIGURE 2. Graphs showing
force-velocity (upper panels)
and force-power (lower panels) data fitted by three different methods. Panels A and
D: Curves fitted to the forcevelocity relation. Panels B and
E: The curves are fitted to the
same data expressed as the
force-power relation. Panels C
and F: The curves fitted to
force-velocity relation weighted
for power. Arrow in panel A
indicates point derived from
record in Figure 1.
u
o
o
s.
6
io
Force
(nN/mn2)
Chiu et al
Effects of Inotropic Interventions in Rabbit Myocardium
1165
TABLE 1. Contractile Properties of Isolated Muscles in the Control State Before Intervention
Intervention
Postextrasystolic
potentiation
Cross- Rest/
sectional twitch
Number
area
force
of
2
ratio
muscles (mm) (mm )
Twitch
force
(mN/
mm 2 )
Relative
Time
Maximum Tension-time Maximum Maximum maximum
to
power
power
velocity
integral
dP/dT
peak
(raW/g)
(msec) (mN/mm2-sec) (mN-sec/mm2)
12
4.22
±0.80
0.43
±0.14
0.048
±0.017
16.5
±4.3
284
±67
105
±38
2.46
±1.05
2.31
±0.72
4.65
±1.70
0.291
±0.051
Acetylstrophanthidin
6
Isoproterenol
6
Caffeine
5
4.35
±1.03
4.37
±1.06
3.94
±0.49
0.48
±0.14
0.39
±0.12
0.33
±0.22
0.041
±0.012
0.048
±0.019
0.049
±0.020
16.1
±4.8
19.0
±4.9
15.7
±6.2
306
±44
285
±95
250
±40
88
±17
129
±44
162
±82
2.65
±1.26
2.87
±1.36
2.03
±1.22
2.27
±0.76
2.77
±0.60
2.64
±0.46
4.50
±1.94
5.76
±1.53
4.86
±1.96
0.287
±0.049
0.318
±0.025
0.326
±0.028
Values are mean±SEM. !_„«, longest length at which the developed force-length relation has a positive slope. Relative maximum
power is the maximum power divided by the isometric force.
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power points, and the extrapolated isometric force
was closer to the measured values (Figures 2B and
2E). These fits consistently lay below the high
velocity points (Figure 2B), such that maximum
velocity was consistently underestimated by a few
percent. A very good compromise fit was obtained
when the curves were fitted to the force-velocity
data weighted for power. The square of the residuals of the force-velocity data (i.e., the squared
difference between the observed and fitted values of
velocity) was multiplied by the observed value of
power, and the fitting routine minimized the square
root of the sum of these values (Figures 2C and 2F).
The curves passed very close to the maximum
power points and to the highest-velocity low-load
points. This technique (Figures 2C and 2F) of fitting
the data to force-velocity curves with velocity
weighted for power was, therefore, used in all
subsequent analyses.
Isometric Twitches
The effects of the four different inotropic interventions are illustrated in Figure 3. Each interven-
tion caused a twofold increase in twitch force, but
the time course of the twitch was affected in different ways. Postextrasystolic potentiation (Figure
3A) and acetylstrophanthidin (Figure 3B) caused
twitch force to increase with only a slight reduction
in the time to peak force. With these two interventions, the general shape of the twitch was little
altered, such that the maximum rate of rise of force
(dP/dtnupi) and the time-tension integral during the
rise of force scaled in approximate proportion to the
peak force (Table 1). By contrast, isoproterenol
caused a substantial reduction in time to peak force
(Figure 3C) so that the rate of rise of force was
increased to a much greater extent than peak force
and the time-tension integral was only slightly
increased. Caffeine had the opposite effect (Figure
3D). Table 2 compares the mean effects of the four
interventions on these three commonly used indexes
of contractility for all muscles in the series.
The effects of the first three interventions were
fairly straightforward and were measured simply
after the addition of the intervention. The effects of
caffeine, on the other hand, were more dependent
on stimulation frequency. When caffeine was given
to muscles stimulated at the usual interval of 1.22.0 seconds, the addition of caffeine caused an
initial large increase in twitch force, with a prolonged twitch, as shown in Figure 3D. This initial
effect usually did not last sufficiently long to measure the force-velocity curves. Within a few min-
TABLE 2. Percent Change in Isometric Twitch Parameters With
Different Inotropic Interventions
TABLE 3. Percent Change In Force-Velocity Properties With
Different Inotropic Interventions
Results
The isometric twitch and force-velocity parameters
for each set of control contractions are listed in Table
1. The effects of each intervention on these parameters is described separately for isometric twitch in
Table 2 and for force-velocity parameters in Table 3.
Intervention
Postextrasystouc
potentiation
Acetylstrophanthidin
Isoproterenol
Caffeine
Maxi- Timemum tension
Number Twitch Time
of
force to peak dP/dT integral
(%)
(%)
(%)
muscles {%)
12
6
6
5
Values are mean±SEM.
140±49 -11+71 194±87 120±69
101±39 -11±5 159±39 108±43
113±29 - 4 0 ± l l 272±79 22±30
78±25 55 ±41 2±8 134±101
Number
Intervention
Postextrasystolic
potentiation
Acetylstrophanthidin
Isoproterenol
Caffeine
Maxi-
Maxi-
mum
of
Twitch mum
muscles force velocity power
12
6
6
5
Values are mean±SEM.
Relative
maximum
power
140±4 9±19 172±60 13±5
101±4 5±24 115±68 5±17
113±8 l±20 157±36 21±19
78±5 2±8 124±42 19±12
1166
Circulation Research
Vol 65, No 5, November 1989
FIGURE 3. Graphs showing comparison of the effects of four interventions on the time course of the
twitch. In each panel the force has
been divided by the peak force in the
control twitch (ref).
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200
400
600
0
TIME Cms)
utes, force fell to about 1.5 times control, and the
time to peak fell to near control levels. The effects
described here were obtained in a later series of
experiments in which the stimulation interval was
increased to 2.0-2.5 seconds. This slowing of the
stimulation rate caused the control twitch force to
fall, but it also allowed the caffeine-mediated increase
of the force and the prolongation of time to peak
force to last for as long as a half hour, which was
much longer than was necessary to collect the data
for a force-velocity curve.
Force-Velocity Relations
The effects of the four interventions on the instantaneous force-velocity relations were measured to
compare the influence of these agents on the contractile machinery. Typical quick-release force and
length records from control and postextrasystolic
records are superimposed in Figure 4. The force in
the potentiated contraction is 2.2 times that of the
control throughout the record, but the relative forces,
that is, the ratio of the isotonic load to the isometric
force immediately before the step, are almost identical for the two. The length records show an initial
rapid shortening that is substantially larger for the
potentiated contraction. This early rapid shortening
is attributed to recoil of elastic elements in series
with the contractile elements, mainly at the ends of
the muscle. This shortening is expected to be larger
in the potentiated contraction because the drop in
absolute force during the step is larger. Once the
force step is complete, the subsequent shortening is
similar in the two contractions.
Quantitative measurements of force and velocity
were made over a 4-msec period between 6 and 10
msec after the release (between the dotted lines in
Figure 4), and the foree-velocity points obtained at
different relative loads in different contractions are
plotted in Figure 5. Figure 5A shows th© velocity
plotted as a function of the absolute force in control
and potentiated contractions. To show how the
curves scale, velocity is plotted as a function of
relative force in Figure 5B. The plots nearly superimpose. The extent to which the curves coincide
after scaling the forces will depend to some extent
on the maximum velocity. If the maximum velocities are not the same, the curves will not superimpose exactly. In the plots of Figure 5, the maximum
velocity for the potentiated contractions is slightly
FIGURE 4. Force and length recordings superimposed
for control and postextrasystolic potentiated twitches.
Lmav longest length at which the developed force-length
relation has a positive slope. The step to the isotonic load
occurred 40 msec earlier in potentiated record, and the
record has been shifted horizontally so that the times of
the tension steps coincide. Muscle length was 4.3 mm;
cross-sectional area was 0.71 mm2.
Chiu et al Effects of Inotropic Interventions in Rabbit Myocardium
3
1167
Po Vnax PViax
Control
1.07 2..76 .36
Potentiated 1.07 2..88 .41
L A
5. Force-velocity (upper panels) and force-power (lower panels) plots
for control andpostextrasystoUcpotentiated twitches. LmMX, longest length at
which the developed force-length relation has a positive slope; Po, isometric
force; Vn^, maximum velocity; PVmax,
relative maximum power; P, isotonic
force. The left panels show velocity and
power plotted as a function of absolute
force. In the panels on the right, force
is plotted as a fraction ofPo. Arrow in
panel B indicates points derived from
records in Figure 4.
FIGURE
_.
IB
*
Y
f
\\
I •t\
i~^«
U
o
12
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u
•\
31
10
Force (nN/m2)
40
0
Relative Force (P/ft)
higher, as are the remainder of the velocity points.
In general, a slight (1-9%) increase in maximum
velocity was obtained with all four interventions
(Table 3). To know whether the velocities at intermediate loads were increased, it would be necessary to measure velocity at some intermediate reference load. The relative maximum power
(maximum power divided by the isometric force)
provides a convenient reference point in the middle
of the curve.22 As with maximum velocity, there is
a slight increase in the relative maximum power in
the example shown (Figure 5D) and in the curves
obtained with all interventions (Table 3). Although
slight (5-21%), these differences are statistically
significant with three of the four interventions and
suggest that the velocities at intermediate relative
loads are definitely, but not substantially, increased.
Comparison of the Effects of
Different Interventions
Table 3 compares the three parameters of the
force-velocity curves for the different interventions.
All four interventions caused an approximately twofold increase in the isometric twitch force. The
small increases in maximum velocities were not
significant and not significantly different from each
other. The increases in relative maximum power
were significant at the/»<0.05 level with three of the
four interventions, but none of the increases were
significantly different from each other.
Discussion
The effects of the inotropic agents on twitch
contractions in rabbit myocardium described here
are similar to those described earlier for cat papil-
lary muscle.1 The main point to be made from the
observations of isometric twitches is that the ranking of inotropic effectiveness depends very strongly
on the index used to measure contractility. If peak
twitch force were used to assess the changes, all
four interventions would have been regarded as
being nearly equally effective. If the maximum rate
of force development is used, isoproterenol is much
more effective, and caffeine is much less so than the
others. The reverse order is obtained with the
time-tension integral; caffeine is the most effective,
and isoproterenol has only a slight positive effect.
These data show that "contractility" can be strongly
dependent on the way it is defined.
The similarity of the effects of all agents on the
force-velocity relation is in marked contrast to the
very different effects of the agents on the time
course of the twitch. These similarities and differences place some limits on the possible sites of
action of the various interventions. These sites of
action can be divided into three broad categories: 1)
calcium release into the myofilament space, 2) calcium binding to the myofilament, and 3) the contractile response of the activated myofilaments.
Each of these will be discussed separately.
Calcium Release
Calcium release has been measured optically by
injecting the calcium-sensitive bioluminescent protein aequorin into cells. All four of the agents
studied have been reported to increase the size of
the calcium transient after stimulation of cardiac
muscle1 although the effect of caffeine is variable
and, in the concentration used here, causes a
decrease. 123 In the control state, the optical signal
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peaks very early in the contraction and declines to
only slightly higher than rest levels shortly after
twitch force reaches its peak.24 There is a substantial lag between the time course of the free calcium
signal and the time course of twitch force. The four
interventions studied here cause changes in the time
course of the calcium signal that parallel to some
extent the time course of twitch tension described
here.1 Increases in stimulus rate and acetylstrophanthidin cause the optical signal to become larger with
little change in the time to its peak. The signal
reaches its peak at about the same time as in control
twitches. Catecholamine causes the optical signal to
reach an earlier and higher peak than in the control
state. By contrast, caffeine causes a prolonged
release of calcium, with the optical signal reaching a
higher peak much later than in the control twitches,
and at the concentrations used here, the peak is
often reduced.1-23
For two of the interventions studied, the changes
seen in the time course of the twitch can be explained
by the observed changes in calcium release. Acetylstrophanthidin and postextrasystolic potentiation
cause an increase in the amount of calcium released
with very little change in the time course of this
release. As a result, the amplitude of the twitch
increases with relatively small changes in time
course. The slightly shortened time to peak force
can be explained by the sarcoplasmic reticulum
sequestering calcium more rapidly when the free
calcium level is higher, such that relaxation begins
slightly earlier.
Caffeine is known to impair calcium accumulation
by the sarcoplasmic reticulum. Once calcium is
released, its reaccumulation is delayed. The higher
forces seen with caffeine can, therefore, be explained,
at least in part, by the greater time for force development. The rise of force is not terminated as quickly by
the reduction of calcium in the myofilament space.
The observation that the peak of the calcium transient
may also be diminished suggests that there may be an
additional explanation.
The effects of isoproterenol are not so easily
explained by the known changes in the calcium
transient because the rate of relaxation is increased.
Therefore, there must be a change in the processes
that intervene between the changes in the free
calcium level and the changes in force. A process
that is likely to be changed is the rate at which
calcium dissociates from the myofilaments.
Calcium Binding to Myofilaments
McClellan and Winegrad25'26 have developed a
chemically skinned cardiac muscle preparation that
can be activated and relaxed by changing the external calcium ion concentration in the presence of
MgATP (see review by Winegrad27). This preparation retains its membrane-bound hormone receptors
so that the contractile response to calcium after
hormonal intervention can be altered. /3-Adrenergic
stimulation decreases the preparation's sensitivity
to calcium; that is, it requires a higher calcium ion
concentration to achieve a given level of partial
force. The decreased calcium sensitivity is associated with phosphorylation of troponin, the calciumbinding protein on the thin filaments. In this preparation, therefore, one effect of /J-adrenergic
stimulation is the lowering of the affinity constant
for calcium binding to the myofilaments.
It seems surprising that calcium binding would be
reduced in the presence of a positive inotropic agent
until it is realized that this reduction is likely to
hasten relaxation. The decreased calcium sensitivity seen with isoproterenol suggests that more calcium must be released to achieve a given increase in
force than is required with the other agents. Much
more calcium must be cycled with each beat. This
increase in calcium cycling requires a larger expenditure of energy, which is in keeping with the
observation that adrenergic stimulation increases
the total energy expenditure of the myocardium and
decreases efficiency.28
The finding that caffeine increases the calcium
sensitivity of skinned cardiac muscle1 suggests
another way that caffeine could prolong twitch
duration and increase twitch force. Both of these
changes would be explained if caffeine decreased
the dissociation rate of calcium from myofilaments.
Contractile Responses of the
Activated Myofilaments
The preceding discussion suggests that all of the
changes in the isometric twitches could be accounted
for by changes in the amount and time course of
calcium binding to the myofilaments. This time
course could be changed either by a change in the
time course of calcium release and reuptake or by a
change in the time course of calcium binding. Both
types of changes can be grouped together as changes
in activation. No change in the contractile response
of the activated myofilaments was needed to account
for the observed differences in isometric twitches.
The force-velocity curves must be examined to see
if it is necessary to postulate any additional changes
in the contractile properties of the muscle.
Some experiments with skinned skeletal muscle
fibers have shown that changes in activation do not
alter maximum velocity but depress velocities at all
finite loads.22-29-30 In addition, force-velocity curves
obtained at different levels of activation can be made
to superimpose if velocity is plotted against the relative rather than the absolute force, as was done in
Figure 5B. It should be mentioned that this is still a
somewhat controversial area (see review by Podolin
and Ford31), but over the range of conditions studied
here (i.e., assessing just the first few milliseconds of
shortening and at levels of activation between maximum and about half maximum), most recent studies
indicate that maximum velocity does not change with
activation.22-32-34 In the present study, the relative
force-velocity curves obtained in different inotropic
states do not superimpose exactly as would be
Chiu et al Effects of Inotropic Interventions in Rabbit Myocardium
expected from the results obtained in skeletal muscle.
In a previous study of the effects of changing activation on the force-velocity properties of cat papillary
muscles,14 a similar result was obtained. Forcevelocity curves were measured at different times in
the twitch and, therefore, at different levels of activation. The relative force-velocity curves superimposed
nearly, but not exactly. The differences in velocity at
each load could either be attributed to a direct effect
of activation on the contractile elements or to a small
internal load.
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Internal Load
In the presence of an internal load, the contractile
elements cannot be fully unloaded, and the true
maximum velocity cannot be achieved. Different
maximum velocities are seen at different levels of
activation because the internal load is a different
fraction of the isometric force. Maximum velocity is
affected more than other parameters used to describe
the curve because the force-velocity curves are
very steep at low loads and small changes in these
low loads can cause large changes in the intercept of
the curves with the velocity axis.
In an earlier study of cat myocardium at different
times in the twitch,14 an internal load equivalent to
6% of the isometric force at the peak of an unpotentiated control twitch could have caused all of the
differences in the relative force-velocity curves.
Such a load would be doubled, to 12% of isometric
force, when activation had risen to only half its
peak value and could have reduced the apparent
maximum velocity by 20%, as was found. In the
present study, in which isometric force was
increased twofold, an internal load equivalent to 4%
of the isometric force would have been reduced to
2% of isometric force, and measured maximum
velocity would have been increased by 5-10%,
approximately what was found here. A constant
internal load will cause the entire force-velocity
curve to shift horizontally. Inspection of the curves
in Figure 5C shows that a leftward shift of the
potentiated curve by 2% of isometric force would
cause the curves to almost coincide. The coincidence would not be exact, but the differences over
most of the curve would be less than 1% of isometric force. The greatest difference would occur in the
high-load low-velocity points. This observation suggests that, if there is an internal load, it may be
shortening dependent; that is, it may be some type
of viscoelasticity, as previously described.11-13
These considerations indicate that the changes in
maximum velocity described here can be accounted
for by a small internal load. They do not, however,
prove that this is the cause of the changes seen. On
the basis of the data presented, it is equally possible
that the changes are due to some other effect of the
potentiating agent on the force-velocity relation. The
important point to be made about these experiments is
that the effects of all of the interventions are the same.
This observation suggests, but does not prove, that
1169
the changes produced by the different agents have the
same effect on the contractile machinery.
Changes in the Contractile Machinery
Force and shortening in muscle is believed to be
caused by the force-generating crossbridge that
forms between the thick and thin myofilaments.35
These crossbridges, which are a structural part of
the myosin thick filaments, cyclically attach to the
actin thin filaments, generate force, and detach. In
generating force, the attached bridges move through
a limited range of motion and stretch a spring
somewhere in the bridge. Changes in the amount of
isometric force can be produced either by a change
in the number of attached bridges or in the average
force per attached bridge, that is, by a shift in the
average position of an attached bridge. Measurements of isometric force alone cannot distinguish
between these two mechanisms. Increases in activation are believed to increase the number of
attached force-generating bridges by making more
of the thin filament attachment sites receptive.
Thus, increases in activation increase the number of
attached force-generating bridges without influencing the average force per bridge. By contrast, an
intervention that changes the average force per
bridge would have to shift the average position of an
attached bridge. Such a shift would be likely to
influence the cycling kinetics of the bridges. Since
shortening velocity is dependent on the cycling rate
of the bridges, an inotropic agent that influences the
average force per bridge would also be likely to
change shortening velocity. This is the reason for
our measuring the force-velocity relation as well as
the isometric force response in the present study.
We chose postextrasystolic potentiation as one of
the interventions because of the belief that its onset
of action is so brief that there would be little time to
produce a change in the chemistry of the contractile
proteins. If this is so, then the effects of other
agents could be compared with this intervention to
see if they differed in some way. The principal
finding in this study was that none of the chemical
agents caused an effect that was different from
postextrasystolic potentiation.
It is likely that some interventions might change
the contractile machinery in a way that would be
manifested as changes in the force-velocity properties. Pagani and Julian,16 for example, have shown
that a change in the myosin type within the muscle
causes profound changes in the force-velocity properties. Cardiac muscle can contain three types of
myosin made up of two types of myosin heavy
chains, a and p. Homodimers, either V, or V3, are
made of two a- or two 0-myosin heavy chains and
have fast or slow ATPase rates, respectively. A
heterodimer, called V2, is made of one a- and one
/3-myosin heavy chain and has an intermediate
ATPase rate. The shortening velocity of the muscle
is strongly dependent on the myosin type within the
muscle. Muscle containing mostly V, myosin short-
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Circulation Research Vol 65, No 5, November 1989
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ens about six times faster than muscle containing
mostly V3 myosin.16
As mentioned above, McClellan and Winegrad
and their colleagues25-26-36 have shown that /3adrenergic stimulation decreases the sensitivity of
the myofilaments to calcium. They have further
shown that /3-adrenergic stimulation increases isometric force at supramaximal levels of activating
calcium and that this increase occurs only in proportion to the relative amount of V, myosin in the
muscle (see review by Winegrad27). Hoh et al37
found that /J-adrenergic stimulation of rat papillary
muscle containing a predominance of Vj myosin
increases the frequency of the minimum in dynamic
stiffness plots, an effect that they interpret as being
due to faster crossbridge cycling. These effects can
be interpreted either as increases of a selective
activation of the fast forms of myosin or of changes
in the contractile kinetics of the fast form of myosin. Thus, it seems quite likely that /3-adrenergic
stimulation could, in some circumstances, increase
shortening velocity to a much greater extent than
postextrasystolic potentiation.
This inability to see a substantial change in shortening velocity with /3-adrcnergic stimulation in the
present study may be due to the preponderance of
V3 myosin in the adult euthyroid animals studied
here.38 Because some types of inotropic interventions can cause a change in the proportion of the
different types of myosin being activated, it is
important that the muscles being studied have a
preponderance of one type of myosin when changes
in the dynamics of the crossbridge cycle are sought.
Comparison With Earlier Studies
The original studies of the influence of inotropic
stimulation on the force-velocity properties of papillary muscle used afterloaded contractions2-4 and
purported to show that maximum velocity was very
strongly dependent on inotropic state. Similar conclusions were reached from studies in which maximum velocity was determined during a period of
continuous shortening at very low loads.39 These
types of studies have been criticized severely
because the measurements were made at different
times in the twitch, at different contractile element
lengths, and after different amounts of shortening.5-7
The present study shows that when all the forcevelocity points are made at the same time in the
twitch and at the same contractile element length,
the maximum velocity varies very little with inotropic state. This observation again suggests that conclusions drawn about inotropic state depend very
strongly on the way the measurements are made.
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KEY WORDS • papillary muscle • heart muscle • inotropic
interventions • acetylstrophanthidin
• isoproterenol •
caffeine
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Comparison of the effects of different inotropic interventions on force, velocity, and power in
rabbit myocardium.
Y C Chiu, K R Walley and L E Ford
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Circ Res. 1989;65:1161-1171
doi: 10.1161/01.RES.65.5.1161
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Copyright © 1989 American Heart Association, Inc. All rights reserved.
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