45
J. Physiol. (1976), 260, pp. 45-53 With 3 text-ftgurem Printed in Great Britain
ENERGETICS OF ISOVOLUMIC CONTRACTIONS OF THE ISOLATED RABBIT HEART
BY R. L. COULSON of Cardiology, Temple University Health Sciences Center, From the Section Philadelphia, Pennsylvania 19140, U.S.A.
(Received 25 November 1975) SUMMARY
1. In the isolated beating rabbit heart the heat production (measured by Dewar-flask calorimetry) and oxygen consumption (measured polarographically) increased in a similar way to force development (assessed as the time integral of left ventricular developed pressure) when the diastolic size of the heart was increased. 2. The energy expenditure of the heart consists of an element which is independent of the force developed and another which varies with the force developed. 3. The calorific equivalent of oxygen in the beating heart when it was provided with pyruvate as a substrate was found to be 20-48 mJ/f1.: 02 at 25° C. 4. The anaerobic metabolism was well below 5 % of the total energy liberation and was constant at all levels of mechanical activity. INTRODUCTION
Several authors have examined the mechanical performance of cardiac muscle in relation to its total energy expenditure, which has been determined by measuring heat production or oxygen consumption. McDonald (1966) and McDonald, Taylor & Cingolani (1966) have examined oxygen consumption in whole hearts with developed tension as the index of performance. Gibbs, Mommaerts & Ricchiuti (1967) and Gibbs & Gibson (1969) have used the myothermic method on papillary muscle under isometric and isotonic conditions with developed tension as the performance parameter. Neill, Krasnow, Levine & Gorlin (1963) used an empirical relationship involving coronary arterial-venous temperature gradient to compare oxygen consumption and estimated heat production in whole pumping hearts. Some years ago Professors J. Grayson and D. R. Wilkie (personal communication) placed isolated beating rabbit hearts into a Dewar-flask
B. L. COULSON calorimeter and measured heat production without regard for mechanical performance. McDonald (1971) working in Wilkie's laboratory repeated this exercise but with the addition of isovolumic developed pressure as a performance index. Coulson & Rusy (1973) have also used this method with modifications developed in Wilkie's laboratory. Since a valid first step toward understanding cardiac energetics in the intact organism has been made with the isolated papillary muscle (Gibbs et al. 1967; Gibbs & Gibson, 1969) it appeared that the next logical step could be taken by examination of energetic parameters in the intact but isolated heart. In the present work the calorific equivalent of oxygen in isolated beating rabbit heart has been measured directly. The energetics of pressure maintenance have been examined by simultaneous calorimetric measurement of heat production and polaragraphic assessment of oxygen consumption of the left-hand limb of the Frank-Starling curve of isolated rabbit heart. 46
METHODS
ApparatuB and preparation. The method of McDonald (1971) was extensively modified and is described in detail elsewhere (Coulson & Rusy, 1973). The method consists of perfusing isolated rabbit hearts retrogradely, via the aorta, in a special Dewar-flask calorimeter. The temperature increment between the perfusate entering and leaving the calorimeter (and hence, the coronary circulation) was measured with a pair of thermocouples connected to a recording nanovoltmeter. This temperature increment together with the perfusion solution flow rate was used to calculate the steady-state heat production rate. Arterial and venous oxygen tension was sampled continuously by two polarographic Po, electrodes, one at the entrance and the other at the exit of the calorimeter; these permitted the oxygen tension gradient across the heart to be measured. The oxygen tension gradient, together with the perfusion flow rate and the solubility coefficient for oxygen, permitted the calculation of steady-state oxygen consumption rate. A liquid-filled latex rubber bag was introduced into the left ventricle through the mitral valve and the pressure developed in the bag during contraction was measured with a recording electromanometer. The time integral of the isovolumic pressure developed was obtained with an analogue re-setting integrator. The heart rate was maintained constant with an electronic pace-maker issuing d.c. shocks of 5 V through a bipolar electrode positioned in the right ventricle. Mechanical performance was varied by injecting or withdrawing liquid from the rubber bag, thereby changing diastolic volume. The perfusion liquid was Krebs-Henseleit solution modified to contain 100 /LM Ca-EDTA (for the binding of trace metals) and 10 mm pyruvate as the only metabolic substrate. The solution was aerated with 95% 02 and 5 % CO2 which maintained a pH of 7-4. The entire apparatus was immersed in a thermostatically controlled water-bath at 250 C. Hearts were excised from 2 to 3 kg rabbits of either sex (anaesthetized with pentobarbitone sodium, i.v., 40 mg/kg). The animals were pre-treated with heparin (500 i.u./kg) to avoid coronary coagulation while the hearts were installed in the apparatus. Procedure. Six to eight sets of measurements were made on each of eleven hearts within the first 2 hr of isolation. Measurements of heat production rate (h, mW) and oxygen consumption rate (q, #sl./sec) were made simultaneously over the course of
MYOCARDIAL ENERGETICS OF RABBIT HEART
47
ten cardiac cycles. The time integral of isovolumic developed pressure multiplied by the heart rate gave the equivalent 'mean' pressure (P, mmHg) maintained by the heart during this period. Coronary perfusion rate and heart rate were held constant throughout experiments but varied slightly from heart to heart. The mean heart rate was 126 ± 0-23 beats/sec (mean ± S.D. of observation, n = 11). Variation was entirely due to determining empirically the rate nearest 1 0 beats/sec which could be maintained by electronic pacing. The coronary perfusion rate was set by adjusting the perfusion pump-rate until at the maximum mechanical performance (apex of the isovolumic pressure-volume curve), an oxygen tension of at least 100 mmHg was maintained in the coronary effluent. The mean perfusion rate was 0 304 + 0 44 cm3/sec (mean ±S.D., n = 11). All eleven hearts were weighed after experiments. The left ventricles were weighed together with their intraventricular septa and averaged 3-64 ± 0-22 g (mean ± S.D.) while the right ventricles averaged 0-85 ± 0-11 g. The water content determined by drying at 600 C for 72 hr averaged 77-7 ± 2 3 % for the left ventricles and 79 4 + 2-0 % for the right ventricles. The volume of the ventricular bag was altered incrementally in order to change the level of mechanical performance. Diastolic volume was not increased beyond the point where mechanical performance failed to increase coincidentally. By making changes in the downwards direction from this point the time required for temperature equilibration after a ventricular volume change was greatly reduced. Maintenance of steady-state performance was shown by unchanging temperature and oxygentension gradients across the calorimeter and therefore across the coronary circulation. If, after mechanical performance had been varied from maximal to minimal and the ventricular balloon re-inflated to again give maximal mechanical performance, the developed isovolumic pressure was equal to or greater than that under comparable conditions at the initiation of the experiment, the data were accepted. If the mechanical performance had begun to deteriorate the experiment was discarded.
RESULTS
Isovolumic pressure-volume relationship Fig. 1 illustrates the pooled results of all eleven experiments. Heat production, oxygen consumption and mechanical performance all exhibit significant (P < 0-01) positive correlation with ventricular volume. Correlation and regression coefficients are given in the legend to Fig. 1.
Anerobic respiration and the calorific equivalent of oxygen Fig. 2 illustrates the pooled results of all experiments expressing total heat production as a function of oxygen consumption. In such a plot the intercept on the heat axis represents anerobic metabolism and the slope corresponds to the calorific equivalent of oxygen. However, since neither parameter was a controlled variable there was no immediate reason to suppose that either of the two possible linear regression functions would be more representative of the relationship between these two parameters than the other. Hence both were derived and reason for selecting one will be discussed later. The correlation coefficient between the parameters of heat production and oxygen consumption (r = 0-98) was highly significant
48 R. L. COULSON (P < 0-001, n = 81). The regression of heat upon oxygen yields an intercept of 2-14 + 0-84 mW (mean + S.E. of intercept, n = 81) and a slope of of 19-75 + 0-43 mJ/,A.: 02 (mean +S.E. of slope). For the regression of oxygen upon heat the corresponding values were 0-73 + 0-04 mW and 20-48 + 0-44 mJ/ul.: 02 .AAr
X
I
U
X X
C
E
A *XXXX x X
XXX
0.
E ._
Xx X XXX~~~
-
24 25
XXX Xx~
_
ixN
4c
x
x
X
xxX 400
'
0
0-75 Isovolumic volume (cm3)
2 811 r C
B X
C
X
C
,=143-0
0 X
x
X
bo
x
x
x
X
xN XXX
2097
-
E
N
E_, 0--
56-8
X
0.
u -
1 50
-
X
>
GJ
X
X
X
0
X
II
I * - .,
0
075 Isovolumic volume (cm3)
1 50
I29 2 0
075 Isovolumic volume (cm3)
Fig. 1. Relationship of energetic parameters to left ventricular isovolumic volume: the eighty-one pooled data points from all eleven experiments are illustrated. The abscissae are left ventricular isovolumic balloon volume (V) expressed in cm3. The ordinates are: A, mechanical performance; B, total oxygen consumption; and C, total heat production. Note the zero suppression on the ordinal scales. The ordinates shown encompass only the range of variation encountered in all experiments. The regression lines are described by the following equations: inmA, P = 7-78 + 1-67 mmHg (means + S.E. of intercept) + V x 29-79 + 2-73 mmHg cm-3 (mean + 5.E. of slope); in B, q = 1-566 ± 0-052 1dl. sec-' + V x 0-672 ± 0-086 Ild. sec-1. cm-3; in C, A = 32-77+ 1-02 mW+ Vx 13-81 ± 1-68 mW.cm-3. The correlation coefficients for the data in A, B and C, respectively, are 0-78, 0-66, 0-68 (n = 81). At the mean volume of 0-559 cm-3 the ordinal mean values are 24-44 mmHg (Pa/133.3), 1-941 ,ul.: 02/sec, and 40-49 mW for A, B and C, respectively.
1-50
MYOCARDIAL ENERGETICS OF RABBIT HEART
49
Energetics of mechanical performance Fig. 3 illustrates the relationship between total heat production and mechanical performance. Significant correlation (P < 0.01) was noted (r = 0-847, n = 81). Inspection of the Figure indicated, however, that the 60 x
x
40
X~~~~~~~~~~~~~~~~~'
Co
-v
0
4
2020
I
0
0
I 2-0
I 1 0
I
30
Oxygen consumption (#1. : 02/sec)
Fig. 2. Relationship of heat production to oxygen consumption: the two alternative linear regression lines (not shown) expressed in terms of heat production (h, mW) as a function of oxygen consumption (q, /tl. sec-' are as follow (mean + S.E. of intercept and mean + S.E. of slope, n = 81): h = 2.14 (±0.84)+19-75 (+0.43) xq(huponq) (1) = 0.73 (+0*04) + 20 48 (+0.44) x q (4 upon h). (2) and The correlation coefficient is 0i98 (n = 81). The intercept on the h axis from either regression is significantly
greater than zero (P
xvxxxtxx x5x
x
s 20_
0
0
X
25 Mechanical performance (mmHg
50
[Pa/I133 3])
Fig. 3. Relationship of total heat production to isovolumic mechanical performance of the left ventricle: mechanical performance (F) was altered by variation of the left ventricular isovolumic volume downwards from the point where mechanical performance was maximal. The correlation coefficient is 0-837, n = 81. The linear regression equation relating heat production (h, mW) to mechanical performance (P, mmHg) is h = 28-84 + 0-68 (mean 5.E. of intercept) + 0-477 ± 0-026 (mean ± 5.E. of slope) x P. The standard error of estimate is 2-23 mWN. Non-linearities were apparent and a least square polynomial test fitting indicated an effective representation of the non-linearities is afforded by the cubic equation: A = 26-02 + 1-299 P -0-0514 p2 + 0-00085 P3. The standard errors of the coefficients are ± 2-17, ± 0-308, ± 0-0134 and ± 0-00018 respectively (n = 81). The standard error of estimate is 1-72 mW. In both the linear and cubic models the intercepts are significantly greater than zero (P < 0-01, n = 81).
Anerobic respiration and the calorific equivalent of oxygen Since neither heat production nor oxygen consumption were controlled variables in these experiments there is a statistical problem of deciding which, if either, of the two possible linear regressions is representative of events. The correlation coefficient is very high (r = 0-98, n = 81) s0 there
51 MYOCARDIAL ENERGETICS OF RABBIT HEART is little difference in assuming all the errors of measurement to be in either parameter. However, there is some reason to believe that the equation derived from the regression of oxygen consumption upon heat production (see Fig. 2) is more representative of actual events. The only source of metabolic substrate available to the hearts in these experiments was their own internal intracellular stores ofglycogen and fat and the 10 mm pyruvate in the perfusate. Since the 10 mm pyruvate was likely a substrate concentration far in excess of any internally stored substrate and venous Po. was never less than 100 mmHg it would be reasonably expected that anerobic metabolism would be minimal. The anerobic intercept of the second regression was the smaller of the two. Regardless of which regression is accepted the anaerobic intercepts are very small. However, both perilously long extrapolations to the heat axis yield intercepts which are significantly greater than zero (P < 0.01) (see Fig. 2). The summary oxidation of pyruvate at 250 C represented thus: (PYRUVATE + 2*5 02-+ 3 C02+ 2 H20 + heat) would produce 20-94 mJ/ful.: 02). If indeed the foregoing is sufficient reason to accept the steeper line as being representative of the metabolic activities of the hearts in these experiments then the inference may be made that the majority of variation in the experimental measurements lay with the monitoring of oxygen consumption.
Energetics of mechanical performance It is possible that at low levels of ventricular mechanical heat. Shortening performance some shortening heat was present (see Fig. 3). As isovolumic volume and hence mechanical performance was increased shortening would be expected to decrease due to better superpositioning of the endocardium upon the ventricular balloon surface. This would account for increased heat cost of developed pressure as low volumes were approached but it would not account for the higher heat cost of mechanical performance at larger volumes. Mechanical performance heat. Smith (1972) has argued that apart from basal metabolism the energy expenditure of working frog and toad skeletal muscle is comprised of two components: one, which is independent of mechanical performance and corresponds to calcium activation energy; and another, which is dependent on mechanical activity and therefore the actin-myosin reaction. Gibbs et al. (1967; Gibbs & Gibson, 1969) made similar observations in rabbit papillary muscle. The present work suggests that there is also a force-independent component of heat production in isolated whole hearts (see Fig. 3). Provision for a force-independent parameter appears justified since a significant performance-independent intercept (P < 0 01) was found (see legend to Fig. 3). Some part of the forceindependent heat in these experiments corresponds to the activities of the
52
B. L. COULSON right ventricular free wall and to the basal metabolism, or resting heat of the hearts and could not be isolated in these experiments. However, the intercepts of heat upon performance regression functions should also contain calcium activation (but not pressure-developing) metabolic heat (Smith, 1972). As alluded to earlier (see isovolumic pressure-volume relationship, above) the smaller value of volume-independence in the performance parameter than in the metabolic energetic parameters may be explained by these non-force-related metabolic activities. Since (see Fig. 3) the change in heat production was not linearly related to the changes induced in pressure development (or actin-myosin interaction) it is concluded that some other factor also changed with the pressure-volume relationship. As there is now good evidence that cardiac muscle does not improve its condition of actin-myosin filament overlap with stretch on the left limb of the pressure-volume curve (Winegrad, 1974), volume or length dependent activation is suggested. Activation heat. An explanation for the non-linearity of the relationship (see Fig. 3), other than that attributable to shortening heat, may be found in the hypothesis that increasing stretch on the left-hand limb of the Frank-Starling curve activates a mechanism allowing more activating calcium to be released. Thus, a more effective actin-myosin interaction from essentially the same filament overlap could be obtained. The increased stoichiometric heat cost of the extra calcium pumping could then explain -a non-linearly increased heat cost of over-all mechanical activity even though the actual heat cost per unit of actin-myosin reaction (mechanical force development) remained constant. The sigmoid character of the calcium concentration-force relationship (Julian, 1971) allows for such a non-linear increase in force development for increase in intracellular calcium release. Recently such a length-dependent calcium release mechanism for regulating force production has been demonstrated for the giant barnacle fibre (Ridgway & Gordon, 1975). 'Resting' heat. None of the foregoing has considered the possibility that non-linear changes in steady-state heat production with mechanical performance may have been related to changes in resting heat production. Changes in resting heat production with resting length or tension have been demonstrated in rabbit papillary muscles (Gibbs et al. 1967), but the changes reported were either insignificant or irregular. In the present experiments the pattern of non-linear change of heat production with mechanical performance was always consistent. I enjoy thanking Professors D. R. Wilkie and J. Grayson for the original impetus for this project, the Heart Foundation and the Medical Research Council of Canada for personal support, and the South-eastern Pennsylvania Heart Association for research funds.
MYOCARDIAL ENERGETICS OF RABBIT HEART
53
REFERENCES
COurLSON, R. L. & Rusy, B. F. (1973). A system of assessing mechanical performance, heat production and oxygen utilization of isolated perfused whole hearts. Cardiovascular Re8. 7, 859-869. GIBBS, C. L. & GIBSON, W. R. (1969). Effect of ouabain on the energy output of rabbit cardiac muscle. Circulation Re8. 24, 951-967. GIBBS, C. L., MOMMAERTS, W. F. H. M. & RICCHIUTI, N. V. (1967). Energetics of cardiac contractions. J. Physiol. 191, 25-46. JULIAN, F. J. (1971). The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J. Physiol. 218, 117-145. McDONALD, R. H., JR (1966). Developed tension: a major determinant of myocardial oxygen consumption. Am. J. Physiol. 211, 667-673. McDONALD, R. H. JR (1971). Myocardial heat production: its relationship to tension development. Am. J. Physiol. 220, 894-900. MCDONALD, R. H., JR, TAYLOR, R. R. & CINGOLANI, H. E. (1966). The measurement of myocardial developed tension and its relation to oxygen consumption during ventricular contraction and relaxation. Am. J. Physiol. 211, 667-673. NEILL, W. A., KRASNOW, N., LEVINE, H. J. & GORLIN, R. (1963). Myocardial anaerobic metabolism in intact dogs. Am. J. Physiol. 204, 427-432. RIDGWAY, E. B. & GORDON, A. M. (1975). Muscle activation: effects of small length changes on calcium release in single fibres. Science, N. Y. 189, 881-883. SMITH, I. C. H. (1972). Energetics of activation in frog and toad muscle. J. Physiol. 220, 583-599. WINEGRAD, SAUL (1974). Resting sarcomere length-tension relation in living frog heart. J. gen. Physiol. 64, 343-355.
J. Physiol. (1976), 260, pp. 45-53 With 3 text-ftgurem Printed in Great Britain
ENERGETICS OF ISOVOLUMIC CONTRACTIONS OF THE ISOLATED RABBIT HEART
BY R. L. COULSON of Cardiology, Temple University Health Sciences Center, From the Section Philadelphia, Pennsylvania 19140, U.S.A.
(Received 25 November 1975) SUMMARY
1. In the isolated beating rabbit heart the heat production (measured by Dewar-flask calorimetry) and oxygen consumption (measured polarographically) increased in a similar way to force development (assessed as the time integral of left ventricular developed pressure) when the diastolic size of the heart was increased. 2. The energy expenditure of the heart consists of an element which is independent of the force developed and another which varies with the force developed. 3. The calorific equivalent of oxygen in the beating heart when it was provided with pyruvate as a substrate was found to be 20-48 mJ/f1.: 02 at 25° C. 4. The anaerobic metabolism was well below 5 % of the total energy liberation and was constant at all levels of mechanical activity. INTRODUCTION
Several authors have examined the mechanical performance of cardiac muscle in relation to its total energy expenditure, which has been determined by measuring heat production or oxygen consumption. McDonald (1966) and McDonald, Taylor & Cingolani (1966) have examined oxygen consumption in whole hearts with developed tension as the index of performance. Gibbs, Mommaerts & Ricchiuti (1967) and Gibbs & Gibson (1969) have used the myothermic method on papillary muscle under isometric and isotonic conditions with developed tension as the performance parameter. Neill, Krasnow, Levine & Gorlin (1963) used an empirical relationship involving coronary arterial-venous temperature gradient to compare oxygen consumption and estimated heat production in whole pumping hearts. Some years ago Professors J. Grayson and D. R. Wilkie (personal communication) placed isolated beating rabbit hearts into a Dewar-flask
B. L. COULSON calorimeter and measured heat production without regard for mechanical performance. McDonald (1971) working in Wilkie's laboratory repeated this exercise but with the addition of isovolumic developed pressure as a performance index. Coulson & Rusy (1973) have also used this method with modifications developed in Wilkie's laboratory. Since a valid first step toward understanding cardiac energetics in the intact organism has been made with the isolated papillary muscle (Gibbs et al. 1967; Gibbs & Gibson, 1969) it appeared that the next logical step could be taken by examination of energetic parameters in the intact but isolated heart. In the present work the calorific equivalent of oxygen in isolated beating rabbit heart has been measured directly. The energetics of pressure maintenance have been examined by simultaneous calorimetric measurement of heat production and polaragraphic assessment of oxygen consumption of the left-hand limb of the Frank-Starling curve of isolated rabbit heart. 46
METHODS
ApparatuB and preparation. The method of McDonald (1971) was extensively modified and is described in detail elsewhere (Coulson & Rusy, 1973). The method consists of perfusing isolated rabbit hearts retrogradely, via the aorta, in a special Dewar-flask calorimeter. The temperature increment between the perfusate entering and leaving the calorimeter (and hence, the coronary circulation) was measured with a pair of thermocouples connected to a recording nanovoltmeter. This temperature increment together with the perfusion solution flow rate was used to calculate the steady-state heat production rate. Arterial and venous oxygen tension was sampled continuously by two polarographic Po, electrodes, one at the entrance and the other at the exit of the calorimeter; these permitted the oxygen tension gradient across the heart to be measured. The oxygen tension gradient, together with the perfusion flow rate and the solubility coefficient for oxygen, permitted the calculation of steady-state oxygen consumption rate. A liquid-filled latex rubber bag was introduced into the left ventricle through the mitral valve and the pressure developed in the bag during contraction was measured with a recording electromanometer. The time integral of the isovolumic pressure developed was obtained with an analogue re-setting integrator. The heart rate was maintained constant with an electronic pace-maker issuing d.c. shocks of 5 V through a bipolar electrode positioned in the right ventricle. Mechanical performance was varied by injecting or withdrawing liquid from the rubber bag, thereby changing diastolic volume. The perfusion liquid was Krebs-Henseleit solution modified to contain 100 /LM Ca-EDTA (for the binding of trace metals) and 10 mm pyruvate as the only metabolic substrate. The solution was aerated with 95% 02 and 5 % CO2 which maintained a pH of 7-4. The entire apparatus was immersed in a thermostatically controlled water-bath at 250 C. Hearts were excised from 2 to 3 kg rabbits of either sex (anaesthetized with pentobarbitone sodium, i.v., 40 mg/kg). The animals were pre-treated with heparin (500 i.u./kg) to avoid coronary coagulation while the hearts were installed in the apparatus. Procedure. Six to eight sets of measurements were made on each of eleven hearts within the first 2 hr of isolation. Measurements of heat production rate (h, mW) and oxygen consumption rate (q, #sl./sec) were made simultaneously over the course of
MYOCARDIAL ENERGETICS OF RABBIT HEART
47
ten cardiac cycles. The time integral of isovolumic developed pressure multiplied by the heart rate gave the equivalent 'mean' pressure (P, mmHg) maintained by the heart during this period. Coronary perfusion rate and heart rate were held constant throughout experiments but varied slightly from heart to heart. The mean heart rate was 126 ± 0-23 beats/sec (mean ± S.D. of observation, n = 11). Variation was entirely due to determining empirically the rate nearest 1 0 beats/sec which could be maintained by electronic pacing. The coronary perfusion rate was set by adjusting the perfusion pump-rate until at the maximum mechanical performance (apex of the isovolumic pressure-volume curve), an oxygen tension of at least 100 mmHg was maintained in the coronary effluent. The mean perfusion rate was 0 304 + 0 44 cm3/sec (mean ±S.D., n = 11). All eleven hearts were weighed after experiments. The left ventricles were weighed together with their intraventricular septa and averaged 3-64 ± 0-22 g (mean ± S.D.) while the right ventricles averaged 0-85 ± 0-11 g. The water content determined by drying at 600 C for 72 hr averaged 77-7 ± 2 3 % for the left ventricles and 79 4 + 2-0 % for the right ventricles. The volume of the ventricular bag was altered incrementally in order to change the level of mechanical performance. Diastolic volume was not increased beyond the point where mechanical performance failed to increase coincidentally. By making changes in the downwards direction from this point the time required for temperature equilibration after a ventricular volume change was greatly reduced. Maintenance of steady-state performance was shown by unchanging temperature and oxygentension gradients across the calorimeter and therefore across the coronary circulation. If, after mechanical performance had been varied from maximal to minimal and the ventricular balloon re-inflated to again give maximal mechanical performance, the developed isovolumic pressure was equal to or greater than that under comparable conditions at the initiation of the experiment, the data were accepted. If the mechanical performance had begun to deteriorate the experiment was discarded.
RESULTS
Isovolumic pressure-volume relationship Fig. 1 illustrates the pooled results of all eleven experiments. Heat production, oxygen consumption and mechanical performance all exhibit significant (P < 0-01) positive correlation with ventricular volume. Correlation and regression coefficients are given in the legend to Fig. 1.
Anerobic respiration and the calorific equivalent of oxygen Fig. 2 illustrates the pooled results of all experiments expressing total heat production as a function of oxygen consumption. In such a plot the intercept on the heat axis represents anerobic metabolism and the slope corresponds to the calorific equivalent of oxygen. However, since neither parameter was a controlled variable there was no immediate reason to suppose that either of the two possible linear regression functions would be more representative of the relationship between these two parameters than the other. Hence both were derived and reason for selecting one will be discussed later. The correlation coefficient between the parameters of heat production and oxygen consumption (r = 0-98) was highly significant
48 R. L. COULSON (P < 0-001, n = 81). The regression of heat upon oxygen yields an intercept of 2-14 + 0-84 mW (mean + S.E. of intercept, n = 81) and a slope of of 19-75 + 0-43 mJ/,A.: 02 (mean +S.E. of slope). For the regression of oxygen upon heat the corresponding values were 0-73 + 0-04 mW and 20-48 + 0-44 mJ/ul.: 02 .AAr
X
I
U
X X
C
E
A *XXXX x X
XXX
0.
E ._
Xx X XXX~~~
-
24 25
XXX Xx~
_
ixN
4c
x
x
X
xxX 400
'
0
0-75 Isovolumic volume (cm3)
2 811 r C
B X
C
X
C
,=143-0
0 X
x
X
bo
x
x
x
X
xN XXX
2097
-
E
N
E_, 0--
56-8
X
0.
u -
1 50
-
X
>
GJ
X
X
X
0
X
II
I * - .,
0
075 Isovolumic volume (cm3)
1 50
I29 2 0
075 Isovolumic volume (cm3)
Fig. 1. Relationship of energetic parameters to left ventricular isovolumic volume: the eighty-one pooled data points from all eleven experiments are illustrated. The abscissae are left ventricular isovolumic balloon volume (V) expressed in cm3. The ordinates are: A, mechanical performance; B, total oxygen consumption; and C, total heat production. Note the zero suppression on the ordinal scales. The ordinates shown encompass only the range of variation encountered in all experiments. The regression lines are described by the following equations: inmA, P = 7-78 + 1-67 mmHg (means + S.E. of intercept) + V x 29-79 + 2-73 mmHg cm-3 (mean + 5.E. of slope); in B, q = 1-566 ± 0-052 1dl. sec-' + V x 0-672 ± 0-086 Ild. sec-1. cm-3; in C, A = 32-77+ 1-02 mW+ Vx 13-81 ± 1-68 mW.cm-3. The correlation coefficients for the data in A, B and C, respectively, are 0-78, 0-66, 0-68 (n = 81). At the mean volume of 0-559 cm-3 the ordinal mean values are 24-44 mmHg (Pa/133.3), 1-941 ,ul.: 02/sec, and 40-49 mW for A, B and C, respectively.
1-50
MYOCARDIAL ENERGETICS OF RABBIT HEART
49
Energetics of mechanical performance Fig. 3 illustrates the relationship between total heat production and mechanical performance. Significant correlation (P < 0.01) was noted (r = 0-847, n = 81). Inspection of the Figure indicated, however, that the 60 x
x
40
X~~~~~~~~~~~~~~~~~'
Co
-v
0
4
2020
I
0
0
I 2-0
I 1 0
I
30
Oxygen consumption (#1. : 02/sec)
Fig. 2. Relationship of heat production to oxygen consumption: the two alternative linear regression lines (not shown) expressed in terms of heat production (h, mW) as a function of oxygen consumption (q, /tl. sec-' are as follow (mean + S.E. of intercept and mean + S.E. of slope, n = 81): h = 2.14 (±0.84)+19-75 (+0.43) xq(huponq) (1) = 0.73 (+0*04) + 20 48 (+0.44) x q (4 upon h). (2) and The correlation coefficient is 0i98 (n = 81). The intercept on the h axis from either regression is significantly
greater than zero (P
xvxxxtxx x5x
x
s 20_
0
0
X
25 Mechanical performance (mmHg
50
[Pa/I133 3])
Fig. 3. Relationship of total heat production to isovolumic mechanical performance of the left ventricle: mechanical performance (F) was altered by variation of the left ventricular isovolumic volume downwards from the point where mechanical performance was maximal. The correlation coefficient is 0-837, n = 81. The linear regression equation relating heat production (h, mW) to mechanical performance (P, mmHg) is h = 28-84 + 0-68 (mean 5.E. of intercept) + 0-477 ± 0-026 (mean ± 5.E. of slope) x P. The standard error of estimate is 2-23 mWN. Non-linearities were apparent and a least square polynomial test fitting indicated an effective representation of the non-linearities is afforded by the cubic equation: A = 26-02 + 1-299 P -0-0514 p2 + 0-00085 P3. The standard errors of the coefficients are ± 2-17, ± 0-308, ± 0-0134 and ± 0-00018 respectively (n = 81). The standard error of estimate is 1-72 mW. In both the linear and cubic models the intercepts are significantly greater than zero (P < 0-01, n = 81).
Anerobic respiration and the calorific equivalent of oxygen Since neither heat production nor oxygen consumption were controlled variables in these experiments there is a statistical problem of deciding which, if either, of the two possible linear regressions is representative of events. The correlation coefficient is very high (r = 0-98, n = 81) s0 there
51 MYOCARDIAL ENERGETICS OF RABBIT HEART is little difference in assuming all the errors of measurement to be in either parameter. However, there is some reason to believe that the equation derived from the regression of oxygen consumption upon heat production (see Fig. 2) is more representative of actual events. The only source of metabolic substrate available to the hearts in these experiments was their own internal intracellular stores ofglycogen and fat and the 10 mm pyruvate in the perfusate. Since the 10 mm pyruvate was likely a substrate concentration far in excess of any internally stored substrate and venous Po. was never less than 100 mmHg it would be reasonably expected that anerobic metabolism would be minimal. The anerobic intercept of the second regression was the smaller of the two. Regardless of which regression is accepted the anaerobic intercepts are very small. However, both perilously long extrapolations to the heat axis yield intercepts which are significantly greater than zero (P < 0.01) (see Fig. 2). The summary oxidation of pyruvate at 250 C represented thus: (PYRUVATE + 2*5 02-+ 3 C02+ 2 H20 + heat) would produce 20-94 mJ/ful.: 02). If indeed the foregoing is sufficient reason to accept the steeper line as being representative of the metabolic activities of the hearts in these experiments then the inference may be made that the majority of variation in the experimental measurements lay with the monitoring of oxygen consumption.
Energetics of mechanical performance It is possible that at low levels of ventricular mechanical heat. Shortening performance some shortening heat was present (see Fig. 3). As isovolumic volume and hence mechanical performance was increased shortening would be expected to decrease due to better superpositioning of the endocardium upon the ventricular balloon surface. This would account for increased heat cost of developed pressure as low volumes were approached but it would not account for the higher heat cost of mechanical performance at larger volumes. Mechanical performance heat. Smith (1972) has argued that apart from basal metabolism the energy expenditure of working frog and toad skeletal muscle is comprised of two components: one, which is independent of mechanical performance and corresponds to calcium activation energy; and another, which is dependent on mechanical activity and therefore the actin-myosin reaction. Gibbs et al. (1967; Gibbs & Gibson, 1969) made similar observations in rabbit papillary muscle. The present work suggests that there is also a force-independent component of heat production in isolated whole hearts (see Fig. 3). Provision for a force-independent parameter appears justified since a significant performance-independent intercept (P < 0 01) was found (see legend to Fig. 3). Some part of the forceindependent heat in these experiments corresponds to the activities of the
52
B. L. COULSON right ventricular free wall and to the basal metabolism, or resting heat of the hearts and could not be isolated in these experiments. However, the intercepts of heat upon performance regression functions should also contain calcium activation (but not pressure-developing) metabolic heat (Smith, 1972). As alluded to earlier (see isovolumic pressure-volume relationship, above) the smaller value of volume-independence in the performance parameter than in the metabolic energetic parameters may be explained by these non-force-related metabolic activities. Since (see Fig. 3) the change in heat production was not linearly related to the changes induced in pressure development (or actin-myosin interaction) it is concluded that some other factor also changed with the pressure-volume relationship. As there is now good evidence that cardiac muscle does not improve its condition of actin-myosin filament overlap with stretch on the left limb of the pressure-volume curve (Winegrad, 1974), volume or length dependent activation is suggested. Activation heat. An explanation for the non-linearity of the relationship (see Fig. 3), other than that attributable to shortening heat, may be found in the hypothesis that increasing stretch on the left-hand limb of the Frank-Starling curve activates a mechanism allowing more activating calcium to be released. Thus, a more effective actin-myosin interaction from essentially the same filament overlap could be obtained. The increased stoichiometric heat cost of the extra calcium pumping could then explain -a non-linearly increased heat cost of over-all mechanical activity even though the actual heat cost per unit of actin-myosin reaction (mechanical force development) remained constant. The sigmoid character of the calcium concentration-force relationship (Julian, 1971) allows for such a non-linear increase in force development for increase in intracellular calcium release. Recently such a length-dependent calcium release mechanism for regulating force production has been demonstrated for the giant barnacle fibre (Ridgway & Gordon, 1975). 'Resting' heat. None of the foregoing has considered the possibility that non-linear changes in steady-state heat production with mechanical performance may have been related to changes in resting heat production. Changes in resting heat production with resting length or tension have been demonstrated in rabbit papillary muscles (Gibbs et al. 1967), but the changes reported were either insignificant or irregular. In the present experiments the pattern of non-linear change of heat production with mechanical performance was always consistent. I enjoy thanking Professors D. R. Wilkie and J. Grayson for the original impetus for this project, the Heart Foundation and the Medical Research Council of Canada for personal support, and the South-eastern Pennsylvania Heart Association for research funds.
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REFERENCES
COurLSON, R. L. & Rusy, B. F. (1973). A system of assessing mechanical performance, heat production and oxygen utilization of isolated perfused whole hearts. Cardiovascular Re8. 7, 859-869. GIBBS, C. L. & GIBSON, W. R. (1969). Effect of ouabain on the energy output of rabbit cardiac muscle. Circulation Re8. 24, 951-967. GIBBS, C. L., MOMMAERTS, W. F. H. M. & RICCHIUTI, N. V. (1967). Energetics of cardiac contractions. J. Physiol. 191, 25-46. JULIAN, F. J. (1971). The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J. Physiol. 218, 117-145. McDONALD, R. H., JR (1966). Developed tension: a major determinant of myocardial oxygen consumption. Am. J. Physiol. 211, 667-673. McDONALD, R. H. JR (1971). Myocardial heat production: its relationship to tension development. Am. J. Physiol. 220, 894-900. MCDONALD, R. H., JR, TAYLOR, R. R. & CINGOLANI, H. E. (1966). The measurement of myocardial developed tension and its relation to oxygen consumption during ventricular contraction and relaxation. Am. J. Physiol. 211, 667-673. NEILL, W. A., KRASNOW, N., LEVINE, H. J. & GORLIN, R. (1963). Myocardial anaerobic metabolism in intact dogs. Am. J. Physiol. 204, 427-432. RIDGWAY, E. B. & GORDON, A. M. (1975). Muscle activation: effects of small length changes on calcium release in single fibres. Science, N. Y. 189, 881-883. SMITH, I. C. H. (1972). Energetics of activation in frog and toad muscle. J. Physiol. 220, 583-599. WINEGRAD, SAUL (1974). Resting sarcomere length-tension relation in living frog heart. J. gen. Physiol. 64, 343-355.
