Effect of Ouabain on the Energy Output of
Rabbit Cardiac Muscle
By C. L. Gibbs and W . R. Gibson
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ABSTRACT
The effect of ouabain, 0.3 /xg/tnl, on the energy output of rabbit papillary
muscles has been examined by a myothermic technique. The experiments were
conducted at two temperature ranges, 19.2° to 22.8°C and 29.0° to 32.2°C,
and both isometric and afterloaded isotonic contractions were studied.
Temperature differences alone caused pronounced physiological changes, the
higher temperature being associated with lower tension-independent heat and
markedly higher active efficiency, external work/(external work + active heat
production). The heat versus tension curve was rectilinear at higher
temperatures but showed upward curvature at lower temperatures. At 19.2° to
22.8°C, ouabain increased the tension-independent heat by 23%, maximum
tension development by 23%, and mean work output by 39%. Ouabain did not
significantly alter the slope of the heat versus tension curve and increased mean
efficiency only slightly. At 29° to 32.2°C, ouabain did not cause any significant
change in the slope of the heat versus tension curve or in mean muscle
efficiency. Ouabain produced significant increases in maximum tension
development, mean work output, and the tension-independent heat. The effects
of ouabain at the higher temperature were examined at two different calcium
levels, 2.5 and 1.25 ITIM. In the isometric studies the effects of ouabain were
independent of the calcium level, and the calcium level itself had no significant
effect on the heat-tension relationship. In the isotonic studies, ouabain
increased work output but more so at the 2.5 ITIM calcium level. Ouabain did
not affect mechanical efficiency at either calcium level but muscle efficiency
was higher at the 2.5 ITIM calcium level. It is concluded that any effects of the
cardiac glycosides on energy expenditure are consequences of their inotropic
actions and do not represent changes in the energy cost of contraction.
ADDITIONAL KEY WORDS
temperature
calcium
heat-tension relationship
myothermic technique
papillary muscle
tension-independent heat
efficiency
• The inotropic actions of the cardiac
glycosides are well established but there is still
considerable controversy over their metabolic
actions (1-6). The myothermic technique has
therefore been used to investigate the effects
of a cardiac glycoside on cardiac energy
production.
The magnitude of the inotropic response
due to a cardiac glycoside depends upon
several factors including temperature, calcium
concentration in the medium, and number of
From the Department of Physiology, Monash
University, Clayton 3149, Victoria, Australia.
This work was supported in part by a Grant-in-Aid
from the National Heart Foundation of Australia.
Received January 20, 1969. Accepted for publication April 28, 1969.
Circulation Research, Vol. XXIV, June 1969
stimuli applied during the drug equilibration
period (7-13). In the experiments described
here, the last of these was kept constant, but
the effects of two different temperatures and
two different calcium levels were studied.
Both isometric and isotonic experiments
were carried out. The isometric experiments
performed at different muscle lengths permitted the determination of the relationship
between developed tension and cardiac heat
production. It was also possible to measure
the tension-independent or activation heat at
muscle lengths where development of active
isometric tension was zero. Afterloaded isotonic contractions were studied to determine
the external work done plus the total energy
output of the preparations over a wide range
951
952
of loads. These two measurements allowed
calculation of the active mechanical efficiency
of the muscles before and after exposure to
ouabain.
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Methods
Papillary muscles were obtained from the right
ventricles of rabbits. They had a mean weight of
7.2 mg and a mean length of 7.1 mm under a
resting tension of 0.5 g. Their mean crosssectional area was 1.01 mm2 and ranged between
0.56 and 1.43 mm2. The muscles were suspended
vertically in a chamber containing 55 ml of KrebsHenseleit solution which was aerated with 95%
O2-5% CO2 and had a pH of 7.4. The first series
of experiments was conducted at room temperature which ranged from 19.2° to 22.8°C, and the
second series was run at temperatures ranging
from 29° to 32.2°C. After dissection, the muscles
were stimulated to contract isotonically under a
0.5-g load. This period of preloaded isotonic
contractions normally lasted at least 2 hours,
during which time there was generally a continual
improvement in contractility with most of the
improvement occurring in the first hour. Eventually a stable state was reached which could be
maintained for 4 to 8 hours. In the changeover
experiments the experimental protocol was a long
one, and for this reason the equilibration period
was reduced to 1 hour and in such experiments
there was a time-of-day effect (see Results).
Mechanical Measurements.—Each preparation
was fixed at both ends with braided non-capillary
silk, and the silk at the ventricular end of the
preparation was clamped 2 mm from the muscle.
The other end of the muscle was connected by a
stainless steel tube to either an isometric
transducer (Sanborn FTA-100) or to an isotonic
transducer (a modified Brush Metripak). When
operating isometrically, the total compliance of
the transducer, stainless steel tube, and cotton
ties was 1.2 X 10"3 cm/g wt. The isotonic lever
had a lever ratio of 20:1, and the equivalent mass
of the lever plus stainless steel tube was 560
mg.
Stimulation.—The preparation was stimulated
by two flexible platinum electrodes cantilevered
from the thermopile frame. The stimulus voltage
was adjusted to be 10% above threshold
(range 3 to 6 v), and the stimulus duration was
0.5 msec at room temperature and 0.3 msec at
temperatures close to 30°C.
Heat Measurements.—The thermopile used
had an output of 3.45 mv/°C and was used in
conjunction with an Astrodata Model 120-nv
amplifier whose frequency response was reduced
to 20 Hz by a filter network. The heat loss from
the muscle thermopile system was practically
GIBBS, GIBSON
exponential. It averaged 11.3%/sec and was
corrected for electrically. The calibration procedure and the corrections to be made for the
stimulus artifact have been described previously
(14, 15). For the experiments at 29° to 32.2°C, a
control vessel (Haake Model N. B. S. Thermostat) was used to circulate fluid to the experimental water bath containing the muscle chamber.
With this system, baseline stability was not a
problem and closely approached the stability
found at room temperature. No attempt has been
made to correct the time course of the heat
records for conduction delays. Because of the
relatively high heat capacity of the thermopiles
and the unknown distribution of adhering fluid,
any such analysis would not be warranted.
Comparative studies of the rate of heat production made under the same physical conditions are
still valid but the heat records cannot be directly
compared with the mechanical records; the
former are undoubtedly too slow. The heat
associated with the contractile response has been
called the fast-phase heat, and there is evidence
that this heat corresponds to the initial heat of
skeletal muscle (16). To measure the total heat
(i.e., initial plus recovery heats) with reasonable
accuracy, it is necessary to measure the total heat
liberated in a train of contractions and then to
divide the summed heat by the number of stimuli
in that train. The experiments can be either
isometric or isotonic ones. The average total heat
value per contraction can then be plotted against
the average developed tension or the average
work done. In the graphs and tables presented in
this paper the measured heat (or energy) is
always the average total heat per contraction. It
should be noted that in afterloaded isotonic
contractions the load is allowed to stretch the
muscle out to its original length. This work done
by the load upon the muscle appears as heat, and
hence the total heat measurement alone gives the
total energy output of the muscle, i.e., external
work and active heat production.
Efficiency.—The problems of interpreting efficiency measurements as they relate to muscle
contraction have been discussed elsewhere (1719). We have used the following definition:
active efficiency =
external work
external work + active heat
external work
~ total isotonic heat'
The reason why the latter formula can be used is
given in the preceding section.
Isometric Contractions.—In experiments involving isometric contractions, tension was altered
by changing muscle length. The relationships
between developed tension (g/cm 2 ) and heat
Circulation Research, Vol. XXIV, June 1969
953
OUABAIN AND CARDIAC ENERGETICS
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production (mcal/g muscle weight) for different
muscles under different conditions were expressed
as polynomials. The polynomials were fitted using
the criterion of least squares, tension being
treated as the independent variable and heat
production as the dependent variable (20).
Analysis of variance was used to find components
of variability attributable to the regression and
due to deviation from regression and to determine
whether inclusion of the higher order terms
reduced the error variance appreciably (21,
chapter 16). In all cases, polynomials of the first
or second degree were considered most suitable.
Isotonic Contractions.—Isotonic contractions
were afterloaded ones. The muscle length under a
resting tension of 0.5 g was taken to be l0, and
the tension generated at this length was called
Po. In this paper the loads are expressed as
fractions of Po found for the prevailing conditions. To simplify the analysis and presentation of
data, we always took values for work, total
energy, and efficiency which corresponded to
certain load values (i.e., 0.0, 0.2, 0.4, 0.6, 0.8,
and 1.0 X P J . Since the loads actually used
were generally not exact fractions of Po, we
plotted graphs of the observed data and read the
appropriate values (e.g., at 0.2 Po) from the
graphs. There were always at least six points on
each graph and frequently double this number.
Experimental Design.—There are now several
reports that the positive inotropic effect of the
cardiac glycosides in mammalian cardiac muscle
is chiefly dependent upon the number of
contractions during the exposure period (13). For
this reason care was taken to standardize the
exposure period to ouabain. In all experiments,
therefore, the muscles were stimulated at a rate of
15/min for 45 minutes before the standard
experiments were commenced. Higher stimulus
rates would probably have raised the percentage
increased in contractility in the presence of
ouabain, but as we were often carrying out
experiments lasting 6 to 8 hours we decided it
was more important not to risk any deterioration
effects caused by depletion of the energy reserves
of the preparations.
Room Temperature.—Results were first obtained without ouabain, the total energy output
being measured in a train of 15 contractions at a
stimulus rate of 15/min. The muscle was then
exposed to Krebs solution containing ouabain, 0.3
yitg/ml, for 45 minutes and the total energy
output measured again as before.
Data were handled using analysis of variance
as for a randomized complete-block design (21,
chapter 8). Each muscle preparation was considered as a block, to which each of the two
treatments was applied once. Thus the variance
attributable to differences between muscle prepCirculaiion Research, Vol. XXIV, June 1969
arations was separated from the variance due to
experimental error.
Temperature 29°C to 32.2°C—These experiments were divided into two groups. In the first
group the same experiments were performed as at
the lower temperature except that the stimulus
rate was increased to one every 2 seconds, and
the total number of contractions in each experimental train was 20. The drug exposure period
remained at 45 minutes and during this time the
preparation was stimulated at a rate of 15/min.
In the second group of experiments the design
was changed in two ways. Firstly, the effects of
two different calcium levels (2.5 mil and 1.25
ITIM) on the action of ouabain were studied, i.e., a
2 x 2 factorial arrangement of treatments was
used (21, chapter 11). Secondly, the treatments
were applied in turn to each muscle preparation
in such a way as to eliminate any confounding of
treatment effects with effects due to temporal
changes in the properties of the preparation, and
to minimize the likelihood that carryover effects
of the drugs would affect the estimates of
treatment effects. A changeover design was used
in which the four treatments were applied to each
of the 8 muscle preparations in a planned
sequence, so that variance due to temporal
changes in the muscles, as well as variance due to
differences between muscles, could be isolated
(22). Thus the experimental material was
arranged in two 4 x 4 latin squares, each having
4 columns (i.e., muscle preparations) and 4 rows
(i.e., positions in the sequence of application of
treatments). A 45-minute stabilization period was
allowed before each treatment.
The data obtained in the first group were
pooled with corresponding data (i.e., data
obtained after 2.5 mM calcium) from the second
group. Analysis of variance was performed as for
a randomized complete-block design.
Presentation of Data.—Significant differences
due to ouabain have been indicated by footnotes
associated with the mean values in the tables.
The P values were determined using analysis of
variance. Due to limitations of space the detailed
analyses have not been presented, but have been
left on deposit.1
Results
ROOM TEMPERATURE
Pilot experiments suggested that a concentration of ouabain of 0.3 fJ-g/ml produced a
reasonable inotropic response over a 45x
For detailed analyses, order document NAPS00422 from ASIS National Auxiliary Publications
Service, c/o CCM Information Sciences, Inc., 22 West
34th Street, New York, New York 10001; remitting
$1.00 for microfiche or $3.00 for photocopies.
GIBBS, GIBSON
954
.26mcol/g
J 52 mcol/g
J .52mcal/g
I 19
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J V
J 5.2 mcol/g
J13 mcol/g
I1"
FIGURE 1
Heat (top traces) and tension (bottom traces) responses
to a single stimulus (a and h) and to a train of stimuli
(c and d). Records a and c were obtained before and
records b and d after treatment with ouabain. In a and
b, the upper heat traces show the tension-independent
heat and the lower traces show the fast phase of active
heat production. The horizontal lines indicate the heat
produced by the stimulus. Time calibration = 1.0
second (a and b) and 20 seconds (c and d).
minute exposure time. Higher doses were tried
(e.g., 1.0 fig/ml), but although the inotropic
response was more evident initially, it could
not be maintained and the preparations would
not last satisfactorily over the time required.
The magnitude of the inotropic response at
the 0.3 fig/ml level varied considerably from
preparation to preparation. Whether this
variability was due to different threshold
sensitivities to the drug has not been investigated. In general, if the tension increment at
l0 after treatment was slight, then there was
relatively little effect of ouabain on the
contractile duration. The classical mechanical
effect of ouabain can be seen in Figure 1. The
upper traces in a and b show the mechanical
response and the heat output per isometric
contraction. This action of ouabain can be
compared with that of epinephrine (see Fig. 2
of ref. 23). The inserts in the heat traces of a
and b show the tension-independent heat
liberated with the muscle shortened to a
length at which no tension was developed.
These traces can be compared with Figure 4
of reference 23. A difficulty about comparisons
of single contractions can be seen by examining traces c and d, where the total heat in a
block of contractions is shown. It has always
been the practice to initiate a single contraction at some fixed time after a preceding train
of contractions. This has been done in an
endeavor to keep the stimulus history of the
muscle as uniform as possible. Notice, however, that ouabain completely alters the usual
staircase pattern (see Fig. 1). Following
treatment with ouabain, the first contraction
after a rest interval is nearly always a maximal
response, whereas under normal conditions, a
maximal response would only occur after
several beats in a train of stimuli. This effect
of ouabain was extremely reproducible at all
temperatures and necessarily complicates any
single beat comparisons. In Figure 2 the time
course of the tension-independent heat in
response to three stimuli is shown before and
after exposure of a preparation to ouabain.
The increment is not as striking as that shown
in Figure 1, but it is still of the order of 40%.
Heat-Tension Relationship.—The method
of obtaining a heat versus tension curve has
been set forth elsewhere (14), but in this
paper the results have been expressed in terms
Circulation Research, Vol. XXIV. June 1969
OUABAIN AND CARDIAC ENERGETICS
955
FIGURE 2
Tension-independent heat responses to three stimuli given under normal conditions (left) and
after ouabain (right). The horizontal lines indicate the amount of heat produced by the stimulus
itself. Heat calibration = 0.35 mcal/g; time calibration = 1.0 second. Muscle weighed 8.2 mg
and was shortened from 7.1 to 5.6 mm to prevent tension development.
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h= .413+ .00390 T + .00000507 T2
h= .509+ .00341 T + .00000514 T2
3.0
-H.2.0
1.0
100
200
TENSION
300
g crrr«
400
500
FIGURE 3
The relationship at room temperature between heat production and tension per cross-sectional
area. The curves represent the regression equations obtained from the pooled data of 16
muscles. The thin line is the relationship obtained before and the thick line that obtained after
exposure to ouabain.
of tension per cross-sectional area of muscle.
By using this type of plot, we hope to make
our results directly comparable with myothermic studies on other types of muscle. The
relationship between heat production and the
tension developed was almost rectilinear in all
muscles, whether ouabain was present or not.
The fitted straight line accounted for more
than 95% of the variation in the observed
values for heat production. However seconddegree polynomials gave a significantly
Circulation Research, Vol. XXIV, June
1969
(P<0.05) better approximation to the observed values in 16 out of 32 cases (i.e., a
significantly smaller residual sum of squares
than remained after fitting a straight line).
Furthermore the second-degree coefficient was
positive in 30 out of 32 cases and was negative
for only one muscle preparation, whether
ouabain was present or not.
The coefficients of the fitted second-degree
polynomials were treated as observations to be
handled by three separate analyses of vari-
956
GIBBS, GIBSON
""HI
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h
J
FIGURE 4
Tension, work and heat production before (top traces) and after exposure to ouabain (bottom
traces). Records a and £ show isometric tension development; tension calibration = 1.0 g.
Other records show distance shortened under loads of 2.0 g (b and g), 1.5 g (c and h), 1.0 g
(d and i), and 0.5 g (e and ]); length calibration = 0.2 mm. Heat calibration on all records =
2.24 g-cm. Muscle weight = 3.1 mg; muscle length = 5.5 mm; temperature = 29°C.
ance, one for each of the three coefficients
needed to describe the curves. By simply
taking the mean value for each coefficient
(data from 16 muscles), the following equations (shown graphically in Fig. 3) were used
to describe the heat-tension relationship: No
ouabain, h = 0.413 + 0.00390T + 0.00000507T2;
ouabain (0.3 /ug/ml), h = 0.509 + 0.00341T
+ 0.00000514T2, where h is the heat produced
(mcal/g) and T is the tension developed
(g/cm 2 ) in a single contraction.
Analysis of variance showed that only the
zero-order coefficient (intercept of the curve)
was affected by ouabain (P<0.05). That is,
apart from the 23% increase in the tensionindependent or activation heat component,
ouabain did not significantly alter the cost of
tension development.
Analysis of the observations made when
tension development was zero showed that
ouabain increased (P < 0.001) the tensionindependent heat from 0.45 to 0.56 mcal/g.
Circulation Research, Vol. XXIV, June 1969
957
OUABAIN AND CARDIAC ENERGETICS
TABLE 1
Effect of Ouabain on External Work Done, Total Energy Expenditure, and Mechanical
Efficiency/ at Room Temperature with Varying Loads (Expressed as Fractions of P o )
P/Po
Drug
0.2
0.0
None
Ouabain, 0.3 Mg/ml
None
Ouabain, 0.3 Aig/ml
0.45
0.56
None
Ouabain, 0.3 ^g/ml
0.4
0.6
External Work (mcal/g)
0.170
0.252
0.238
0.246
0.354
0.329
0.150
0.204
Total Energy (mcal/g)
1.24
1.77
2.11
1.62
2.24
2.59
2.33
2.74
13.1
14.6
Efficiency (%)
13.7
11.0
15.1
12.0
Overall
mean
1.0
1.8
0.203
0.283*
2.39
2.81
1.71
2.09*
6.2
6.9
11.0
12.2*
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Division of table values for external work by corresponding values for total energy does not
yield precisely the efficiency values shown in the tables. This is because the efficiency values for
individual muscle preparations have been used to obtain the mean efficiencies shown in all the
tables, and for the analysis of variance.
Means for 16 preparations are given. * Differs significantly (P < 0.001) from the value obtained in the absence of ouabain.
Isotonic Contractions.—In the isotonic studies, the external work performed, the total
energy liberated, and the mechanical efficiency were measured or calculated (see Discussion). Typical experimental traces are shown
in Figure 4. Results for the external work, total
energy liberated, and mechanical efficiency
are given in Table 1, and are shown
graphically in Figure 5. The load has been
expressed as a fraction of the peak isometric
tension at lo. This value (Po) differed
(P < 0.001) according to whether ouabain
was present or not. Ouabain increased the
mean value of Po from 3.4 to 4.2 g/cm2.
Differences between muscle preparations
(blocks) accounted for a large part of the
variation in all three measured parameters.
Ouabain increased (P < 0.001) the average
external work done by 39%. The heat output
per contraction also rose so that the total
energy output rose (P<0.001) by 22%. There
was a small (11%) increase (P<0.001) in
efficiency. These increases occurred at all
loads (the load X ouabain interaction was not
significant).
TEMPERATURE 29°C TO 32.2°C
Heat-Tension Relationship.—It has been
shown that raising the environmental temperature decreases the tension-independent
heat. In Figure 6 a typical heat versus tension
relationship is shown. Notice that the heat
TABLE 2
Effect of Ouabain on Coefficients of Fitted Polynomials Relating .Heat Production to Tension
Developed during a Single Contraction at 29° to 32.2° C
Second-degree polynomials
Straight lines
No ouabain
Ouabain, 0.3 Mg/ml
Zero-order
coefficient
(intercept)
0.311
0.337
First-order
coefficient
Zero-order
coefficient
(intercept)
First-order
coefficient
Second-order
coefficient
0.00433
0.00466
0.277
0.310*
0.00399
0.00388
0.00000201
0.00000223
Means for 13 muscle preparations are given.
value obtained in the absence of ouabain.
Circulation Research, Vol. XXIV, June 1969
* Differs significantly (P < 0.05) from the
958
GIBBS, GIBSON
2.0
19.5°C/
1/4 sec,
1.5
Ml
I
0.5
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0
1.0
2.0
3.0
TENSION(q)
FIGURE 6
Plot of heat against tension at 19.5"C (solid circles)
and 29.5°C (crosses). Muscle weight = 6.9 mg; muscle length = 6.2 mm.
1.0
FIGURE 5
Results at room temperature. Work, total energy, and
efficiency plotted against P/P o , where P = load and
P o = isometric tension at l0 under the test conditions.
These results are set out in detail in Table 1. Each
point represents the mean of 16 determinations. The
bottom curves indicate normal conditions and the
top curves the presence of ouabain.
versus tension plots obtained at high temperature are more rectilinear. Indeed at higher
temperatures second-degree polynomials gave
significantly (P<0.05) better fits than a
straight line in only 6 out of 26 cases, and in
two of these the second-degree coefficient was
negative. The data for heat-tension relationships at 29° to 32.2°C are shown in Table 2,
the coefficients both for fitted straight lines
and second-degree polynomials being given. A
12% increase (P<0.05) in the tension-independent heat due to ouabain was detectable
only if second-degree polynomials were fitted.
Ouabain caused no significant difference in
the fitted straight lines and no consistent
change in slope or curvature of the seconddegree polynomials.
However, analysis restricted to the observations made with zero tension development
showed that ouabain increased (P < 0.01) the
tension-independent heat from 0.28 to 0.32
mcal/g.
Isotonic Contractions.—The peak isometric
tension at l0 (Po) was increased (P<0.001)
from 2.79 to 3.51 g/cm2 by ouabain, and the
average work output rose (P<0.001) by 27%.
Total energy output rose (P<0.001) by a
similar amount overall, but the increase was
more marked at greater loads than at smaller
ones (ouabain X load (linear) interaction;
P<0.05). Thus, in contrast to the results
obtained at room temperature, efficiency was
not affected by ouabain at a temperature of
Circulation Research, Vol. XXIV. June 1969
OUABAIN AND CARDIAC ENERGETICS
959
TABLE 3
Effect of Ouabain. on External Work Done, Total Energy Expenditure, and Mechanical
Efficiency at 29°C to 32.2°C with Varying Loads (Expressed as Fractions of Po)
P/Po
Drug
0.2
0.0
None
Ouabain, 0.3 /ig/ml
None
Ouabain, 0.3 lig/ml
0.282
0.322
None
Ouabain, 0.3 ^g/ml
0.4
0.6
0.8
External Work (mcal/g)
0.164
0.236
0.227
0.213
0.279
0.283
0.165
0.230
Total Energy (mcal/g)
1.012
1.247
0.732
1.252
1.586
0.883
1.439
1.855
19.8
22.2
Efficiency (%)
20.7
16.3
21.1
16.3
10.0
10.6
1.0
Overall
mean
0.198
0.251*
1.606
2.086
1.053
1.331*
16.7
17.5
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Means for 13 preparations are given. * Differs significantly (P < 0.001) from the value obtained in the absence of ouabain.
29°C to 32.2°C (Table 3). It is important to
note however, that the increase in temperature
(and possibly rate) has caused mechanical
efficiency to increase markedly above its room
temperature value.
EFFECT OF CALCIUM LEVEL AND OUABAIN AT
29° TO 32.2°C2
The factorial arrangement of treatments
allowed us to test for any interaction between
the calcium level and ouabain, as well as for
their independent effects. The allocation of
these treatments to the different muscle
preparations in a changeover pattern (22)
allowed isolation and measurement of any
changes in the contractile state of the muscles
with time of day.
Heat-Tension Relationship.—Second-degree
polynomials provided significantly (P < 0.05)
better fits to the observed data than did
straight lines in only 6 out of 32 cases. Seconddegree coefficients were negative in 13 out of
32 cases (including 2 of the 6 mentioned
above), in contrast to the almost invariant
presence of upward curvature seen in the
room temperature data.
2
It should be noted that data reported in this section
are not independent of those reported above because
results obtained from these preparations when 2.5
niM calcium was present were pooled with similar
data from 5 other preparations to give the figures
shown in Tables 2 and 3.
Circulation Research, Vol. XXIV, June 1969
Data for fitted straight lines and seconddegree polynomials are shown in Table 4.
Ouabain increased (P<0.05) the intercept
by 12% but did not change the slope or
curvature of second-degree polynomials. The
effect of ouabain did not appear as a
difference in intercept if straight lines were
fitted, but rather as an increase (P < 0.01) in
the slope of the lines. However, analysis
restricted to the observations made when
tension development was zero showed that
ouabain increased (P < 0.01) the tensionindependent heat from 0.34 to 0.39 mcal/g.
The effects of ouabain were independent of
the calcium level, and the calcium level itself
had no significant effect on the heat-tension
relationship. The period of the day (row) did
have an effect (P < 0.05) on curvature, but
the effect is obscure since the first and third
periods tended to have low second-degree
coefficients whereas the second and fourth
periods tended to have high ones.
Isotonic Contractions.—The peak isometric
tension at l0 (Po) was 24% higher in the
presence of 2.5 mM Ca2 + than in the presence
of 1.25 mM Ca 2+ (P<0.001). Ouabain increased (P < 0.001) Po almost equally at both
calcium levels (average 21%).
Results for the external work done, total
energy liberated, and mechanical efficiency
are given in Table 5 and are shown graphical-
GIBBS, GIBSON
960
Effect of Calcium Level and Ouabain on Coefficients of Fitted
Developed at 29° to 32.2°C
Polynomials Relating Heat Production
Str;light lines
Zero-order
coefficient
(intercept)
to Tension
Second-degree polynomials
First-order
coefficient
Zero-order
coefficient
(intercept)
First-order
coefficient
Second-order
coefficient
Calcium, 2.5 mil
Calcium, 2.5 HIM
No ouabain
Ouabain, 0.3 ^g/ml
0.337
0.318
0.00431
0.00492
0.348
0.368
0.00412
0.00412
0.00000000
0.00000103
Calcium, 1.25 n a
Calcium, 1.25 nui
No ouabain
Ouabain, 0.3 ^g/ml
0.305
0.360
0.00428
0.00495
0.304
0.362
0.00411
0.00467
0.00000181
0.00000188
Combined data
for both calcium
levels
No ouabain
0.321
0.00430
0.326
0.00411
0.00000121
Ouabain, 0.3 Mg/ml
0.339
0.00493*
0.365f
0.00440
0.00000175
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The four treatments were applied to each muscle preparation in a planned way so that effects due to order of application
(row effects) could be isolated (see text). Means for 8 muscle preparations are given. Means significantly affected by ouabain
are indicated as follows: *P < 0.01, \P < 0.05.
0.001) by ouabain and also was higher
( P < 0.001) in the presence of 2.5 mM Ca2 +
than in the presence of 1.25 mM Ca2 + . These
effects were additive (ouabain X calcium
interaction neglible). Both were more marked
at higher loads than at lower loads (ouabain
X load [linear] and calcium X load [linear]
interactions were both significant; P<0.05).
ly in Figure 7. Work output fell (P<0.001)
when the muscle was bathed in 1.25 mM Ca2 + .
Furthermore, ouabain increased work output
by only 10% at this calcium level whereas the
increase was 27% when 2.5 mM Ca 2+ was
present (ouabain X calcium interaction;
P<0.05).
Total energy expenditure was raised (P <
Effect of Calcium Level and Ouabain on External Work Done, Total Energy Expenditure, and Mechanical Efficiency at
29°C to 32°C with Varying Loads (Expressed as Fractions of Po)
P/Po
0.0
0.2
0.4
0.6
0.8
1.0
Overall
mean
External Work (mcal/g)
Calcium,
Calcium,
Calcium,
Calcium,
2.5
2.5
1.25
.1.25
Calcium,
Calcium,
Calcium,
Calcium,
2.5
2.5
1.25
1.25
HIM
No ouabain
Ouabain, 0.3 jig/ml
No ouabain
Ouabain, 0.3 Mg/ml
Calcium,
Calcium,
Calcium,
Calcium,
2.5 mM
2.5 mM
1.25 mM
1.25 mM
No ouabain
Ouabain, 0.3 /jg/nil
No ouabain
Ouabain, 0.3 iig/ml
HIM
mM
HIM
mM
No ouabain
Ouabain, 0.3 Mg/ml
No ouabain
Ouabain, 0.3 *ig/ml
0.222
0.280
0.169
0.213
0.310
0.368
0.257
0.312
0.308
0.384
0.291
0.315
0.230
0.326
0.239
0.212
1.020
2.046
1.437
1.692
1.885
2.378
1.617
1.884
0.269
0.341
0.239
0.263
Total Energy {mcal/g)
m.M
mM
mM
0.355
0.391
0.318
0.384
0.935
1.124
0.822
1.028
1.301
1.613
1.157
1.425
2.084
2.639
1.738
2.039
1.363
1.69S
1.181
1.408
Efficiency (%)
22.0
24.3
18.8
20.2
22.4
21.9
19.9
21.3
17.5
17.9
18.2
17.9
11.4
12.5
12.7
10.8
18.3
19.1
17.4
17.0
The four treatments were applied to each muscle preparation in a planned way so that effects due to order of application
(row effects) could be isolated (see text). Means for 8 muscle preparations are given. Statistically significant differences
which are relevant are indicated in the text.
Circulation Research, Vol. XXIV, June 1969
961
OUABAIN AND CARDIAC ENERGETICS
^ra 0.4
1 0.3
DC
|
a:
0.2
0.1
LU
t—
x
LU
0
2.5
02
0.4
0.6
0.8
I
0.2
I
0.4
I
0.6
I
0.8
0.2
0.4
0.6
0.8
1.0
r
g 2.0
I 15
.
LU
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5
i.o
_l
I 0.5,
0
1.0
1.0
FIGURE 7
Results at high temperature. Work, total energy, and efficiency plotted against P/P o as in Figure
5. Results obtained with a calcium level of 1.25 mia are shown in column A and residts with a
2.5 mM calcium level are shown in column B. Each point is the mean of 8 determinations.
These results are shown in Table 5. The bottom curves indicate normal conditions and the
top curves the presence of ouabain.
Ouabain did not affect mechanical efficiency, but the high Ca2+ level did result in
higher efficiency, especially when the load was
small (calcium X load [linear] interaction;
P<0.05).
A feature of major interest was that all three
parameters did vary with time of day (row
effect; P < 0.01), all being low during the first
period of the day. The probable reason for
this result has already been outlined in the
Circulation Research, Vol. XXIV, June
1969
Methods. It is encouraging to find that in the
subsequent periods the mechanical response
remained relatively uniform even over experimental times as long as 8 hours.
Discussion
PHYSIOLOGICAL IMPLICATIONS
Before examining the effects of ouabain
treatment it is necessary to discuss in some
detail the physiological results reported in this
962
GIBBS, GIBSON
2.5
obtained at the higher temperature, relates to
the shortening of the duration of the active
state that occurs under these conditions. For
skeletal muscle, Hill (28) has already postulated the existence of a weak positive
feedback whereby tension prolongs activity
and activity prolongs tension with the consequent liberation of extra energy. It might be
expected that such a feedback system would
be more noticeable in terms of energy output
at temperatures where the duration of the active state is normally much prolonged. Physiological procedures so far analyzed at 37°C have
failed to show that alterations in duration of
the active state can influence oxygen consumption, and it has been suggested that this
is because the cardiac active state is so brief at
this temperature (27). Alternatively, one
might consider whether tension development
is the primary parameter to which energy
expenditure is related. It may be important
that the same experimental data that give a
curvilinear heat versus tension plot at room
temperature can produce a rectilinear heat
versus tension-time integral plot (unpublished
results). In the present series of experiments
we did plot for several muscles curves of heat
versus tension and heat versus the tensiontime integral. The results obtained with and
without ouabain are shown for a typical
muscle in Figure 8. This is not to suggest that
the tension-time integral is a better index of
cardiac oxygen consumption than peak tension
development or contractile element work but,
as Sandberg and Carlson (29) have pointed
out, there are theoretical reasons why the
amount of ATP split in a contraction should
be proportional to the time integral of force.
This problem of determining which mechanical variable correlates best with energy output has stopped us from attempting to analyze
our data in greater detail. Shortening heat for
example has been measured elsewhere by
subtracting the external work from a total
energy plot and comparing the residual with
the energy expected on the basis of a heat
versus tension plot (14). If other variables
such as intensity or duration of the active state
or the tension-time integral are determinants
2.0
1.5
"3
u
J 1.0
Ui
I
.5
0
0.5
2.0
1.0
1.5
TENSION(g)
2.0
2.5
3.0
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1.5
•S 1.0
0
0.5
1.0
1.5
AREA(g.sec)
FIGURE 8
Plots of heat against tension (top) and heat against
area (bottom) obtained on the same muscle before
(solid circles) and after (open circles) ouabain administration. The muscle weighed 4.6 nig and was 6.0 mm
long under a resting tension of 0.5 g. Temperature =
19.5°C.
paper. The statistical analysis used has allowed us to document for the first time the
relationship that exists between tension development and production of active cardiac heat.
Interestingly, this relationship which is normally curvilinear at room temperature becomes rectilinear as the temperature is raised
so that it resembles the relationship between
oxygen consumption and tension development
in whole-heart experiments at 37°C (24-27).
The reason for this change with temperature is
not readily apparent, but it is not because we
have examined different ranges of tension
development. The tension range was kept
approximately constant by increasing the
stimulus frequency at the higher temperature.
One possible explanation for the linear plot,
Circulation Research, Vol. XXIV, June 1969
OUABAIN AND CARDIAC ENERGETICS
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of cardiac oxygen consumption, then the
previous analysis may not be valid. Indeed
preliminary experiments when analyzed using
the tension-time integral as an index of energy
output almost eliminate a shortening heat
component.
The tension-independent heat component or
activation heat has been examined in some
detail recently. The results reported here
confirm that the magnitude of this component
decreases with temperature. It is about 0.25
mcal/g/contraction at 37°C, and this value is
close to that estimated on the basis of oxygen
consumption studies of whole hearts (26,
30).
In our experiments, a temperature rise has
apparently produced an increase in active
efficiency from about 14% at room temperature
to about 21% at 30°C. It is possible to see why
this has occurred if we examine the work done
with, for example, a load equal to 0.4Po. Now
if PO = 3.4 g (20°C) and PO = 2.8 g (30°C)
and if the respective work outputs with loads
equal to 0.4Po are 0.252 and 0.236 mcal/g,
respectively (see Tables 1 and 3), and if we
let Po = 3.4 g be equivalent to a crosssectional tension of 500 g/cm2, then, using the
experimentally determined relationship between heat and tension:
h = 0.413 + 0.00390T + 0.00000507T2 (20°C)
h = 0.277 + 0.00399T + 0.00000201T2 (30°C),
it can be calculated that at room temperature
the mechanical efficiency would be 15.1%
([0.252/(0.252 -I-1.421)] X 100) whereas at
30°C mechanical efficiency is 19.2% ([0.236/
(0.236 + 0.990)] X100).
Now the above calculation is very approximate, but it can be seen that the efficiency
increases because (1) roughly the same amount
of external work is done even though Po (in
spite of the rate increase) is lower at the higher
temperature, (2) there has been a decrease in
the size of the tension-independent heat, and
(3) the slope of the heat versus tension curve
is more steep at low temperatures (see Fig.
6). The increase in efficiency at high temperature that we have found appears to be at
variance with data reported by Reisman and
Circulation Research, Vol. XXIV, June 1969
963
Van Citters (31). These authors reported an
efficiency increase when the temperature was
dropped from 37°C to 27°C. We cannot
explain this discrepancy at present but it may
relate to the different temperature ranges
used, to the different preparations, or to the
inclusion of the resting oxygen consumption in
the whole animal efficiency studies.
EFFECTS OF OUABAIN
There is no doubt that ouabain increased
both the magnitude and the rate of production
of the tension-independent heat. The increase
in magnitude was more obvious when a
separate analysis was made of this component.
It has already been shown that an increase in
the external calcium level or the action of an
inotropic agent such as epinephrine increases
both the rate of production and the magnitude
of this component (32). These effects are
always associated with an increase in the
maximum shortening velocity of the contractile element. Our results show that there is an
interaction between calcium and ouabain
supporting many previous reports in the
literature. Several authors have postulated the
existence of a pool of tissue calcium that
becomes exchangeable during the contractile
response, and they suggest that the positive
inotropic action of the cardiac glycosides is
associated with increased calcium release from
such an area (11, 33). Langer has shown that
development of active tension is proportional
to the calcium content of a region within the
myocardium represented by a specific, kinetically defined phase of calcium exchange and
that a fall in temperature from 30°C to 20°C
results in an increase in the calcium content of
phase 2 accompanied by a positive inotropic
response (34, 35). There is evidence that
phase 2 is probably the sarcotubular region of
the muscle which functions to release calcium.
Langer (36) has argued that a "sodium
pump" lag leads to the increase in phase-2
calcium, and there is already abundant
evidence that cardiac glycosides can cause
inhibition of the sodium pump (37-39). Thus
the effect of ouabain upon the tensionindependent heat component indirectly supports the idea that at least one of the actions
964
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of the cardiac glycosides is upon the excitationcontraction coupling mechanism.
It is quite evident that ouabain has not had
a large effect upon the heat versus tension
relationship except the effect on tensionindependent heat. This immediately poses a
question. Why is tension generation so much
more costly in the presence of epinephrine
which produces almost identical mechanical
effects to those of ouabain? Unfortunately the
heat versus tension curve in the presence of
epinephrine is difficult to measure accurately
because it is continually changing (32). Even
so, it would appear that epinephrine increased
the magnitude of the tension-independent
heat much more than ouabain did. In part
this may be due to differences in drug
concentrations but even in some experiments
with ouabain at a concentration of 1.0 /tig/ ml
the increment was never as high as that seen
in the presence of epinephrine (see Fig. 7 of
ref. 32). A major metabolic difference between these two agents would be the ability of
epinephrine to increase the formation of cyclic
3'5'-adenosine monophosphate, and it has
already been suggested that the level of cyclic
AMP may be one of the factors controlling the
magnitude of the tension-independent heat
component (23).
Until recently it has been customary to state
that cardiac glycosides increase cardiac efficiency (4, 40-42). The experimental basis for
this belief has been that an increased work
output can be obtained from failing hearts
without much change in oxygen consumption.
Studies on intact animals or with whole-heart
preparations are complicated by several factors, and in particular, we would like to draw
attention to the resting oxygen consumption.
A clinical symptom of cardiac failure is an
increased end-diastolic volume of the ventricles due to an increased end-diastolic pressure.
One of the consequences of cardiac glycoside
therapy is a fall in this pressure so that the
dilated heart returns towards its normal size.
Now several authors have reported that the
resting metabolism of the heart is sensitive to
diastolic fiber length (14, 43-45). These results
are not universally accepted (46) but the
GIBBS, GIBSON
evidence is such that experiments on whole
hearts should be designed to determine
whether any increase in oxygen consumption,
consequent upon the inotropic action of
ouabain, is masked by a decrease in the
resting oxygen consumption level. In the
present investigations we have not examined
the effects of ouabain upon resting heat
production. The main reason for this omission
is that there is a continuous fall in the resting
heat rate during the day. This fall occurs even
though there is little or no change in cardiac
contractility over this same period of time. We
can state, however, that for the concentration
of ouabain that we employed there was never
any evidence that ouabain altered resting heat
production. Lee et al. (4) reported that in the
presence of ouabain the active oxygen consumption of isolated papillary muscles rose by
150%, while there was a 600% rise in contractility which occurred some time before the
change in oxygen consumption. We feel that
the heat-tension relationship that we observed
may help explain this increase in mechanical
efficiency. To achieve such an increment in
tension, one must at the outset have a
preparation that is developing a low crosssectional tension, let us say some 50 g/cm2. If
we examine a heat versus tension curve (see
Fig. 3), it can be seen that a sixfold increase
in tension would lead only to about 150% rise
in energy expenditure, whether ouabain was
present or not, i.e., there is normally an
increase in efficiency whenever tension development increases in the sense that the tensionindependent heat becomes a smaller fraction
of the active heat production. The possibility
that the increase in contractility precedes the
increase in energy expenditure was not
examined in our experiments but we would
like to point out that in the experiments of Lee
et al. (4) the oxygen electrode was some
distance from the papillary muscle, and we
think that the possibility must exist that the
time separation between the inotropic and
oxygen consumption effects might be due to
chamber geometry. This conclusion is supported by the results of Coleman (47) and
Klaus and Krebs (48) who found no time lag
Circulation Research, Vol. XXIV, June 1969
OUABAIN AND CARDIAC ENERGETICS
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between the inotropic effect and the increase
in oxygen consumption.
There have been two recent studies (47,
49), one on the intact canine heart and the
other on isolated cat papillary muscle, that
have shown that cardiac glycosides decrease
cardiac efficiency. In the canine heart experiments, heart rate, stroke volume, and mean
aortic pressure were held constant but oxygen
consumption rose by about one third. In
experiments with cat papillary muscle it was
shown that oxygen consumption rose by about
30% in isotonic and isometric contractions and
that if the muscles were arranged to develop
the same tension, by altering muscle length,
tension generation was more costly in the
presence of acetylstrophanthidin. In both
papers the increased oxygen consumption was
attributed to the increase in the velocity of
myocardial shortening produced by the glycosides (50). There is not necessarily a serious
discrepancy between the results reported by
those authors and results presented in this
paper. If the increase in the magnitude of the
tension-independent heat component can be
much greater than we found, then the results
would be quite comparable. The magnitude of
any such increase might depend upon several
factors, such as dose level, animal species,
temperature, or stimulus pattern. It is quite
possible that the magnitude of the tensionindependent heat component may be related
to velocity of fiber shortening, but whether
this is a cause-and-effect relationship remains
to be established.
We conclude therefore that under our
experimental conditions the cardiac glycosides
do not produce major changes in the efficiency
of cardiac muscle and that any increase in
oxygen consumption is associated with and is
probably due to the action of ouabain on the
ability of cardiac muscles to manifest tension
or to lift a load; this latter action is exerted
predominantly on the excitation-contraction
coupling mechanism.
Acknowledgment
We would like to thank Miss D. Harrison for
preparing the illustrations.
Circulation Research, Vol. XXIV, June 1969
965
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Circ Res. 1969;24:951-967
doi: 10.1161/01.RES.24.6.951
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