Comp. Biochem. Physiol.. 1975. |bL 52A. pp. 435 to 440. Pergamon Press. Printed in Great Britain
MYOCARDIAL EFFICIENCY IN THE ISOLATED VENTRICLE OF THE SNAIL, HELIX P O M A T I A L. J. P. HEROLD Laboratoire de Physiologie Animale, Facult(~ des Sciences et des Techniques, La Bouloie, 25030 Besancon Cedex, France (Received 25 September 1974)
Abstraet--l. Mechanical power measurements of snail perfused isolated ventricle were made with increasing hydrodynamic pressures of haemolymph or physiological salines. 2. Microcalorimetry, using the Calvet apparatus, gives the total energy flux: E(-AH) where H is enthalpy, produced by the isometric beating heart. 3. The value of ~t = [mechanical power]/[E(-AH)] is a measurement of myocardial efficiency; it varies from 0"08 to 0.26 in haemolymph or Ringer saline. 4. Efficiency is not affected by lowering PO2 in haemolymph but is modified by increasing K ÷ o r C a 2+ in isotonic salines. INTRODUCTION
(1954) that the hydrodynamic measurements are necessary. Thus, we were able to get closer to the actual work of the organ by using volumetric measurements on the ventricle perfused with hemolymph (Herold, 1966). However, the contraction activity of the heart only represents a part of the total energy brought into play by the myoeard. Only physical measurements of the quantity of heat released at the time of contraction can, under precise conditions, give an evaluation of the results of the energetic processes of the biological system i.e. Y_,(-AH) where H represents enthalpy. However, the technical problems of measuring the heat production of the beating heart are even more complex than when using ergometric measurements. The linear thermo couples developed by Hill (1931) on the skeletal muscle can not be applied to the heart which contracts in three spatial dimensions. There, once again, the answer is to use papillary muscles or cardiac strips which can be assimilated to linear elements and then investigated using the thermo-couples technique (Ricchiuti & Gibbs 1965; Grayson et al., 1971). However, the microcalorimeter techniques developed by Calvet & Prat (1963) are, as Boivinet (1971) pointed out, perfectly suited to the study of biological phenomena having a low production of heat over a long period. Thus, instead of trying to make an approximation of the thermic process of a contraction it is possible to explore the variations in thermic rate of the heart over a period of many hours and thus obtain the integration of energetic production of the myocard. This microcalorimeter technique, developed by Boivinet & Rybak (1969) on a frog heart and by Herold & Cudey (1972) on a snail heart, has shown that the good reproducibility of the thermograms can give probable correlations between the mechanical activity and heat production which take into account the total energetic production of the myocard.
THE STUDY of the energetic characteristics of the isolated heart requires firstly a good knowledge of the mechanical work of the organ and secondly a reproducible measurement of the total energy flux of the isolated biological system. In both cases the difficulties are considerable due to the fact that the heart is a complex mechanism with a low survival rate. With both vertebrate and invertebrate animals the best approach to cardiac work depends on a prior choice of measuring techniques. Thus, Siess et al. (1970) when working on the isolated guinea pig, evaluated the work using the product of the force by displacement at known frequencies expressed in g/cm per hr. To do this they used a displacement transducer of the isotonic lever type. Burns et al. (1972) working on a dog heart, decided to measure the volume variations with a micro-manometer placed in the left ventricle and then connected to a ratemeter, This experiment enabled them to evaluate the isovolume and isotonic work in g/cm 2 per cm. However, if this technique takes into account the anatomical characteristics of the hollow heart muscle it can only be applied to one of these cavities. Due to these inherent difficulties some authors have further developed the Fenn (1923) technique for normal striated muscle and preferred to continue their research on cardiac strips or papillary muscle fragments assimilated with linear elements (Whalen 1961; Strauer, 1973). In the case of a heart with a more simple structure such as that of molluscs the problem is the same except that the survival of the organ is easier to maintain. This work can be measured with the mechanoelectric transducer R.C.A. 5734 which gives values for the force-displacement product in relation to frequency. This transducer was used by Paul (1961); Hill & Schunke (1967) and Almqvist (1973) on the Helix heart and gave an estimation of the dynamic properties of the organ. However, the linear displacement recorded under these conditions did not take into account the contraction of the transversal and oblique MATERIALS A N D METHODS fibers of the heart wall. The hearts came from an homogeneous batch of winter So as to take into consideration the true and full work of the heart we believe, like Schwartzkopff snails (Helix pomatia L.) that the laboratory received in 435
436
J . P . HEROLD
November. These snails were room at temperatures between was between 17-5 and 22 g and heart was 28.6 _ 1-9 rag. Their 79-84 to 83.18~o.
then kept in a darkened 7 and 12°C. Their weight the average weight of the water content varied from
I. Measurement o f ventricle work Part of the shell was taken off at the level of the heart. An incision was made in the pericardium, then another in the auricula so as to push the oval end of the straub canula (l mm diameter) back to the inside of the ventricle cavity. A ligature was made at the level of the auriculoventricle junction and the organ was fixed onto the canula. The aortic end was then ligatured. The section of the auricular strip and the aorta was then free. The isolated heart fixed on the canula became part of a volumetric system with constant pressure. One end of a polyethylene tube fitted perfectly onto the perfusion canula. The other end was linked to a horizontal semi-capillary tube graduated in microliters. A liquid index (coloured water with a lubricator added) moved in the tube at each ventricle systol. The volume measured between the index limits displaced at each cardiac revolution represented exactly what entered the ventricle. The advantage of this system is twofold: its great simplicity and its very low inertia. One variable which can be modified at will is available. This is the height of the perfusion liquid in the canula and this means that conditions of stable and known activity can be imposed. So as to remain within the physiological conditions this parameter can only be increased within reasonable proportions. When going from 2 to 8 cm the ergometric characteristics of the heart are considerably modified: above this there appears to be a dissociation of the contraction of the different muscular zones of the organ. To fulfill the requirements set out by Assendelft et al., 1973, who established the units of the International System (S.I.) recommended in Physiology, we will express the results in power units:watt. As well the advantage of using the International System means that the values of mechanical power can be compared to the total power measured by the microcalorimetric technique. The mechanical power of the isolated ventricle is calculated as follows: P = I.t.g.h.V.N
/~ =
g I1
=
N=
specific weight of the perfusion liquid in kg/m 3 i.e. for hemolymph: 1.013.103 acceleration of the weight 9.81 m/s: height of the perfusion liquid in the canula in m systolic volume read off in ttl. i.e. m 3 . 10 -9 frequency in systols per min. To remain in the physiological values and not to use fractions of systols we have kept the frequency in systols m n - ~ instead of expressing them in Hertz (systols per sec). This is the same as calculating the power in J. s-~ i.e.W.
P is then expressed in watts 10 -6. The measurements are taken 30 min to an hour after preparing the hearts when their automatic activity is stable and regular. 2. Measurement o f ventricle heat production A complete study of the Calvet microcalorimeter is available in "Recent progress in microcalorimetry" Calvet & Prat (1963) and in "Biochemical microcalorimetry" Brown (I 969). The electrical value is measured by the electromotive force (e.m.f.) of the thermo-piles which is proportional to the energy flux between two units. The e.m.f, is measured by the deviation A of a galvanometer which at each moment is proportional to the flux. We have insisted on the use of a differential apparatus (Herold & Cudey, 1972) so as to obtain reproducible ther-
mograms. It is the association in opposition of two microcalorimetric elements having the outer cell at the same temperature 0~,. If 0~,.~ is the temperature of the inside cell of the first element, the thermocouples supply an e.m.f.: ER = n Z(01,.i - 0~) the e.m.f, corresponds to the second element at a temperature of 0in.2 E 2 .= n Y-(0in.2 - 0,.~) the results of the two opposing piles is E = E I - E2
= n E ( 0 i n . i - - 01n.2 )
This procedure makes it possible to eliminate by compensation the parasite phenomena disturbing the measurements on the condition that the two microcalorimetric cells receive the same energy at the same time. There is an advantage: if the external temperature variations are the same for the two cells the e.m.f, result is independent of these variations and the stability of the experimental zero is assured. This was the case in our experiments when the two ceils received an equal and constant flow of gas which assured a stable P O , which was necessary for the survival of the hearts. The calorific power developed by the biological material in the measurement cell was then obtained by the basic Tian equation: w=_PA g where P is the leakage coefficient of the microcalorimetric element in watts per degree. g is the sensitivity constant of the galvanometer in meters per degree. The equation P/g is thus a power divided by a length and it is expressed in watts per meter. It also defines the sensitivity of the calorimeter. Our apparatus had a Pig = 4.90.10 .3 w/m. The experimental accuracy is of the order of 0-8~ The detection threshold in watts is 0.2.10-6 watt i.e. a fifth of a microwatt. A is the deviation of the galvanometer recorder. Thus, in our experiment, W is a power expressed in watt 10 -6" The laboratory cell which takes the ventricles has a volume of 10ml. It was thus not possible to perfuse one or more ventricles. To be sure of having a sufficient heat flow and to obtain accuracy we used at one time l0 ventricles functioning isometrically and stretched on a metal frame. A tension of between 150 and 650 mg was selected at the beginning of the experiment. The frame with the hearts was put into hemolymph or physiological saline which was maintained at a steady PO_, with a gas bubbling (mixture O2-N2) or air P O , = 150 Torr (40 ml/hr per cell). The control cell contained the same volume of saline and received the same gas flow. The opposing cells thus eliminated the parasitic effects due to the expansion of gas or to the vaporization of the liquid in the cells. The reading of A was taken after the initial thermic stabilization i.e. I hr after the cells had been put in the calorimeter. The base reference line or the experimental zero was obtained at the end of the experiment with the injection of a 10~o formol solution which eliminated all thermogenesis.
RESULTS T h e contractile activity o f the heart, its m e c h a n i c a l p o w e r d e v e l o p e d a g a i n s t a n i m p o s e d pressure, only r e p r e s e n t a p a r t o f the total activity that is b r o u g h t into play by the m y o c a r d . O t h e r energetic a s p e c t s were linked to m e t a b o l i c
Myocardial efficiency in isolated ventricle of the snail exchanges such as phosphorylations, decarboxylations, deaminations, and synthesis phenomena. The energy linked to the maintenance of the ionic gradient with its bioelectric consequences also played a part. What is interesting about the microcalorimeter is that it measures the result of all these processes without taking into consideration the intermediary metabolism. At one moment t the deviation of the galvanometer recorder represented the total energy developed by the biological system i.e. E ( - A H ) where H is the enthalpy. It was thus possible to compare the values of the mechanical power with the total power, this relationship represented the efficiency of the biological system. We adopted the same efficient relation as Gibbs et al., 1967: mechanical power 0~.~-
437 /
1 / l
/
PO2 "15 torr : y ,0-937 x + 6.536
J
/" ~ hernolyml~
f
/
Cag÷:y • 1x+8.720
/
./
//"
4~ PO:z,fSO tort : y • 0-755 x+ 6. 213 /~inger : y-0"780 x+ 6 - 0 3 8
,oh/
5
Z(-AH)
1. Determination of the efficiency of the active heart in hemolymph (a) Mechanical power. When the ventricle was perfused with hemolymph at PO2 constant at 150 Torr, the work developed by the organ increased in relation to the height h of the liquid in the perfusion canula. The statistical analysis of the results showed that there is a positive linear correlation (r = +0'992) between the volume of the ventricle systolic volume V fy = 8.9) and the mechanical power P (y = 3.56). It can be represented: y = 0-755 Z + 6"213 (b) Total power. The total energy developed by the heart and measured with the Calvet microcalorimeter at PO_, = 150 Torr was obtained on isometric beating organs submitted to tensions that increase from 150 to 650 rag. It must be mentioned that as a non-stretched heart does not give any mechanical power it gives an energy of 5-8 ___ 1-2.10 - 6 watt which corresponds to the rest metabolism of the organ. (c) Calculation of the efficiency coefficient of ~t. The accuracy of results of the two types of measurements could be considered to be satisfactory, but the expression of ~ remained approximate because the experimental conditions are different. A critical examination shows: (a) the mechanical power measurement was done on hearts perfused with hemolymph at variable pressure (p.g.h.)
I
5 P,
I0 w a t t I 0 "e
Fig. I. Linear correlation between the ventricle systolic volume V, and the mechanical power P of the isolated ventricle perfused with hemolymph (at two values for PO2) and with isotonic saline solutions, equilibrated Ringer solution and enriched with K + or Ca 2+. (b) The total power measurement was done on hearts under variable tensions The pressure-tension correspondance must be adjusted beforehand from a known correlation between the systol frequency and the pressure or tension. Thus, a pressure corresponding to h = 2.10 -2 or a tension of 150 mg imposes a frequency of 32 _ 4 systols m n - t on the heart; whereas for h = 8.10 -2 or 650 mg the frequency is 53 + 5 systois m n - t. The comparison of ergometric and microcalorimetric results is thus possible, but only gives the expression of an approximate efficiency. In the case of an active isolated heart in contact with hemolymph the efficiency relation varies between 0"08 and 0"26. These results can be compared with those obtained by Bing & Michal (1959) using a rat heart where ct = 0.20-0.25 and by Reissman & van Citers, 1959 using the same preparation where ct = 0.07-0.30 according to the frequency of the heart.
2. Factors having an effect on efficiency (a) PO2 of the biological saline. During previous research we have shown that the PO2 of the hemolymph is a factor limiting the heat production of the
Table 1. Mechanical power of an isolated ventricle perfused with hemolymph. Results with standard error, and number of experiments ( ) h.m
10 -2
V.m a . 10 -9
N. systoles mn- i P.watt l0 -6
2__.0"1 6"8 _+ 0'5 34 -+ 3 0.76 _ 0.21 (12)
2___0"1 7.3 -I- 0"4 37 _+ 4 1.78 _ 0.18 (12)
6_+0"1 9'6 -+ 0"5 42 _+ 4 3.99 -+ 0.15 (12)
8_+0'1 11'9 -+ 0'6 49 _+ 5 7.71 _+ 0'16 (11)
Table 2. Total power of the active isolated ventricle in hemolymph Tension mg Z(-AH) watt. l0 -~
150 9.3 4- 0'82 (6)
300 16'5 -I- 1-4 (6)
500 19.2 _ 2'3 (5)
650 29-9 _ 3.4 (5)
438
J.P. HEROLD
I0-
L~ O x 13 4-
Hemolymph P02,15tocr HemOlymph POz-150~rr Rin lit
x
CO~ K~
÷
.'o_ O
x+
5-[3
0
&,
x+
a 04+
~
o
oI
I
IO
I
3O
20
Z (-L~H),
I 40
wott I0 -6
Fig. 2. Graph of the values of the efficiency relations ct
mechanical power = P total power: Z( - AH)
or-Equilibrated physiological saline of the Ringer type. This solution contains in m M / l : NaCI: 73. KCI:
of the ventricle isolated under different conditions: in contact with either hemolymph or isotonic saline solutions. Table 3. Mechanical power and total power of a ventricle isolated at PO2 = 150 Torr P watt 10-* E ( - A H ) watt x
10 - 6
0-76 9-3 0'08
1'78 3.99 7.71 16.5 19.2 2 9 . 9 O"11 0.21 0.26
isolated heart (Herold & Cudey, 1972). Thus, if the enthalpy of the biological system is affected by the PO2 it can be thought that the efficiency relation also depends on it too. The mechanical power and the total power are measured at PO_, = 15 (2~ 02 in N2) maintained constant during the experiment. This value of PO_, was chosen for two reasons: it corresponds to the physiological values in oivo in the plumonary vein of the intact snail; and it is close to the PO250 of the hemocyanine at 20°C i.e. 12 Torr (Redfield, 1934). Under these conditions the comparison of the ergometric and the calorimetric results give the following efficiency values: Table 4. Mechanical power and total power of an isolated ventricle at PO2 = 15 Torr in Hemolymph P watt 10 - 6 5"(-AH) watt 10-6 :t
0'51 6.1 0"08
(b) Composition of the physiological saline. To determine the influence of the ionic composition of the physiological saline on the energetic characteristics of the ventricle we used a Ringer solution as a reference solution as the mineral composition is very similar to that of hemolymph (Burton 1965; Meincke, 1972). Then to show the respective role of the K + and Ca 2 ÷ ions we made enriched solutions of each of these ions and maintained the isotony with NaC1 so that the heart would maintain its automatic properties. It is known that the K ÷ C a 2+ ions have marked antagonistic effects on the mechanical activity of the isolated heart (Jullien, 1936). The problem is to determine if the cardiac efficiency is changed when the ionic relationship is experimentally modified.
1.39 3'29 6.19 12.3 1 4 . 6 21.7 0'11 0"23 0"28
It was found that the work and the total power are lower than those given by the active ventricle at 150 Torr. However, the efficiency relation is hardly different from that obtained under much better oxygenation conditions. This clearly shows that the myocard keeps a metabolism intact with very low oxygenation rates done in vitro.
4. CaCI2: 10.8. pH = 7.2-7-6 and maintains the automatism of the ventricle for many hours. When perfused with this solution the isolated ventricle has the following mechanical performances (Table 5). In this case a positive linear correlation was found between the volume of ventricle systolic volume and the mechanical power. The correlation was y = 0'780Z + 6-038 (r = 0'998) and showed that in this equilibrated solution the mechanical characteristics of the ventricle were very similar to those measured in hemolymph, Compared with the total power obtained using the microcalorimetric technique the efficiency relationship varies in relation to the strain that the ventricle undergoes within the same limits as those defined in hemolymph. Table 6. Mechanical power and total power of an isolated ventricle in ringers solution (PO2 = 150 Torr) P watt 10-6 5"(-AH) watt 10-~'
0.88 2.68 5.7 11-06 12.7 1 7 . 4 2 5 - 4 41.76 0.06 0.15 0'22 0.26
fl-Calcium enriched solution without potassium. A concentrated solution of CaCI2 = 15. NaCl = 72.8 mM/1 maintained the automatic activity of the isolated heart. The mechanogram gave a reduced frequency of 4 0 ~ in relation to the Ringer solution accompanied by a reduced diastolic tonus. However, the amplitude of the systols was increased ( + 2 5 ~ ) . The results coincide with those of Paul (1961) which underlined the fundamental difference of the Helix heart response to calcium in relation to the response of the vertebrate heart where an excess of this ion brings about a systolic arrest. The linear correlation between the volume of the systolic volume and the mechanical power gave y = l Z + 8.720 (r = +0.988). Compared with the total power the mechanical power modified the efficiency relation.
Table 5. Mechanical power of the isolated ventricle perfused with ringer solution (PO2 = 150 Tort) h.m 10-2 V . m 3 10 - 9
N.systoles.mn -t P.watt 10-6
2___0.1 6'6 _ 0-4 41 _ 3 0.88 _ 0'18 (9)
4+0.1 8"2 -I- 0"3 50 _ 4 2-68 + 0.16 (10)
6-1-0.1 10"6 _ 0"4 55 _ 4 5-7 _ 0.16 (10)
8-1-0.1 14'6 4- 0'5 58 _ 4 11.06 -t- 0-14 (10)
Myocardial efficiency in isolated ventricle of the snail Table 7. Efficiencyof the ventricle in a solution enriched with Ca -'+ (15 mM CaCI2, 72-8 mM NaCI) P watt 10-6 Z(-AH) watt 10-6 ~t
0'56 1.53 3.48 5.75 4.6 5'2 9'3 16.6 0'12 0.29 0.37 0'34
The ventricle performance is thus increased with the presence of Ca -'+ although the work and total power values were respectively decreased by an excess of Ca-"+. This phenomena, which was accompanied by a hyperpolarization of the muscle fiber (Burton & Loudon, 1972)was however accompanied by a better efficiency. ~-Potassium enriched solution without calcium. The solution used contained KCI = 8, NaCI = 79-7 mM/l. A mechanical activity of weak amplitude was maintained (-30%), but the frequency was high (+25%). The diastolic tonus was increased (+ 40%) in relation to the Ringer solution. The linear correlation between the volume of the ventricle and the mechanical power gave y = 0.843;( + 4-319 (r = +0.994). The ventricle efficiency was reduced in relation to the balanced solution. Table 8. Efficiencyof the ventricle in a solution of 8 mM KCI and 79.7 NaCI P watt 10-* E(-AH) watt 10-* ~t
0'57 2 ' 1 8 5 ' 3 2 8'57 6.9 19'2 2 6 ' 8 44.8 0'08 fill 0 ' 1 9 0"19
In this case the total power is higher than that measured in the equilibrated solution, whereas the work was considerably decreased. The could be explained by the onset of a cellular alteration brought about by the solution. Elekes et al. (1973) have shown that ultrastructural alterations of the sarcotubular cisternae in the myocardial cells incubated in a solution without Ca 2÷ take place. However, if the efficiency was low, the total power increase in relation to the equilibrated solution suggests that the overall metabolisms were activated. This hypothesis would seem to be confirmed by the fact that under these same conditions the myocardial respiratory exchanges were increased (Herold, 1966) and the excitability of the tissues was increased as the depolarization of the myocardial fibers would prove (Burton & Loudon, 1972).
439
The myocardial efficiency relation can be estab-. lished when it is submitted to different strain conditions. The results show that the efficiency can vary from 0'06 to 0'26 when the ventricle is in contact with hemolymph or a well balanced mineral solution. A decrease of PO-' in the hemolymph does not modify the efficiency although the work and total power are both affected. However, a modification of the ion balance brings about efficiency variations. The K ÷ and Ca -'+ ions have antagonistic effects if both reduce the mechanical power, the enthalpy variation is increased by K ÷ and reduced by Ca 2÷. The measurement of ~ ( - AH) can give an estimation of the tissue excitability. SUMMARY
Work of isolated ventricle of a Mollusc is measured by using perfusion techniques. It is possible to measure the total mechanical power due to all muscular element activity, whatever their spatial arrangement is. The Calvet's microcalorimeter gives, with a good approximation, the total energy flux (~(-AH), where H is enthalpy) resulting from the different energetic aspects: isometric work and metabolism of the biological system. The technical difficulty is to obtain reproducible thermograms when ventricles are in standard conditions of tension and oxygenation. This is possible by means of differential records in two opposite microcalorimetric elements. Then, myocardial efficiency can be obtained under different tensions. Results show that efficiency increases with strain from 0"06 to 0'26, whatever the medium is: hemolymph or equilibrated physiological saline. Lowering the PO2 in hemolymph does not affect myocardial efficiency, although work and total energy are affected. When the ionic balance is altered, efficiency becomes modified: K + and Ca -'+ have antagonistic effects, the two ions lower the mechanical power, but total energy flux is increased by K + and lowered by Ca -'+. Measurements of E ( - A H ) can estimate myocardial excitability. Acknowledgements--I would like to express my thanks to Professor J. Ripplinger who started me off on this work. My thanks also go to G. Cudey whose help with the microcalorimeter was invaluable and to M. Nicolet who looked after the statistical analysis.
CONCLUSION In an isolated Mollusc ventricle the work was measured on the perfused organ and this way it was possible to measure the mechanical power resulting from the activity of all the muscular elements whatever their spatial arrangement were. The Calvet microcalorimeter gave a good approximation for the enthalpy variation resulting from the different energetic aspects, isometric work and metabolic exchanges of the biological system. The technical difficulty was to obtain reproducible thermograms when the ventricles were placed in precise conditions of tension and oxygenation. This is possible due to the differential recordings from the two opposite microcalorimetric elements.
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ALMQVISTM. (1973) Dynamic properties of stretch-uaduced chronotropy in the isolated heart of the snail Helix pomatia. Acta. physiol, scand. 87, 39A-40A. ASSENDELFrVAN O. W., MOOKG. A. & ZIJLSTRAW. G. (1973) International System of Units (S.I.) in Physiology. Pfliigers Arch. ges. Physiol. 339, 265-272. BXNGR. J. & MICHALG. (1959) Myocardial efficience. Ann. N.Y. Acad. Sci. 72, 555-558. BROWN H. D. (1969) Biochemical Calorimetry. Academic Press, New York. BOIVINET P. (1971) Contribution de la Calorimc;trie aux Recherches Biologiques Act,elles. (Edited by BRODAE., LOCKER A. & SPRINGER-LEDERERH.), First European Biophysics Congress. Vienna. Austria.
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BOIVINETP. & RYaAK B. (1964) Etude microcalorim6trique du fonctionnement 61ectrom6canique du coeur de grenouille. L/fe Sci. 8, part. If, l 1-20. BURNS J. W. & COVELL J. W. (1972) Myocardial oxygen consumption during isotonic and isovolumic contractions in the intact heart. Am. J. Physiol. 223, (6) 14911496. BURTON R. F. & LOUDON J. R. (1972) The antagonistic actions of calcium and magnesium on the superfused ventricle of the snail Helix pomatia. J. Physiol., Lond. 220, 363-381. CALVET E. '~" PRAT H. (1963) Recent progress in Microcalorimetry. Academic Press. New York. ELEKES K., KISS T. & KATALIN S-ROZSA. (1973) Effect of Ca-free medium on the ultrastructure and excitability of the myocardial cells of the snail, Helix pomatia L. J. molec. Cell. Cardiol. 5, 133-138. GIBaS C. L., MOMMAERTS W. F. H. M. & RICCHIUTI N. V. (1967) Energetics of cardiac contractions, d. Physiol., Lond. 191. 25--46. GRAVSON J., COULSONR. L. & WINCHESTER B. (1971) Internal calorimetry, assessment of myocardial blood flow and heart production. J. appl. Physiol. 30, (2) 251-256. HEROLD J. P. (1966) Consommation d'oxyg6ne et travail du coeur isol6 d'Escargot Helix pomatia L. C.r. Soc. Biol. 160, (7) 1442-1445. HEROLD J. P. & CUDEV G. (1972) La PO2: facteur limitant de la thermog6n6se du coeur de I'escargot. J. Physiol., Paris, 65, (3) 423 A. HEROLD J. P. & ConEY G. (1972) Approche de 1'6nerg6tique cardiaque d'un Mollusque par la microcalorim6trie. C.r. Soc. Biol. 166, (4/5) 561-564. HILL A. V. (1931) Myothermie experiments on the frog's gastrocnemius. Proc. R. Soc. B. 109, 267-303. HILL R. B. & SCHUNKE P. J. (1967) Contractility cycle of an isolated gastropod ventricle. Experientia 23, 570, 1-6.
JULLIEN A. (1936) Des r6actions compar~es des coeurs de Vert6br6s et d'Invert6br6s vis h vis des ~lectrolytes et des drogues. Th~se Bailliere J. B., Paris. MEXNCKE K. F. 0972) Osmotischer Druck und ionale Zusammensetzung des Hiimolymphe winterschlafender Helix pomatia bei Konstanter und sich zyklisch iindernder Temperatur. Z. vergl. Physiol. 76. 226-232. PAUL O. H. (1961) Effects of calcium on the spontaneous contractions of the isolated ventricle of the snail Helix pomatia. Experientia, 17, (7) 310-311. REDFIELD A. C. (1934) The Haemocyanins. Biol. Reos. Cambridge. Phil. Soc. 9, 175-212. REISSMAN K. R. & VAN CITERS R. L. (1959) Oxygen consumption and mechanical efficiency of the hypothermic heart. J. appl. Physiol. 9, 427-430. RICCmUTI N. V. & Gmas C. L. (1965) Heat production in a cardiac contraction. Nature N.Y. 5013. 897-898. SCHWARTZKOPFF J. (1954) Uber die Leistung des isolierten Hertzens der Weinbergschnecke (Helix pomatia L.) im Kiinstlichen Kreislauf. Z. vergl. Physiol. 36, 543-594. SIESS M., KELLER H. J., SCHARE E. & GEISSLER J. (1970) The continuous and simultaneous measurement of 0 2consumption, rate of decarboxylation of ~4C-substrates and the performance of spontaneously-beating isolated heart atria of Guinea pigs. J. molee. Cell. Cardiol. 1, 261-269. STRAUER B. E. (1973) Force-velocity relations of isotonic relaxation in mammalian heart muscle. Am. J. Physiol. 224, (2) 431-434. WHALEN W. J. (1961) The relation of work and oxygen consumption in isolated strips of cat and rat myocardium. J. Physiol., Lond. 157. 1-17.
Key Word Index--Helix pomatia; isolated ventricle; perfusion; mechanical power; Calvet microcalorimetry; enthalpy; efficiency.
MYOCARDIAL EFFICIENCY IN THE ISOLATED VENTRICLE OF THE SNAIL, HELIX P O M A T I A L. J. P. HEROLD Laboratoire de Physiologie Animale, Facult(~ des Sciences et des Techniques, La Bouloie, 25030 Besancon Cedex, France (Received 25 September 1974)
Abstraet--l. Mechanical power measurements of snail perfused isolated ventricle were made with increasing hydrodynamic pressures of haemolymph or physiological salines. 2. Microcalorimetry, using the Calvet apparatus, gives the total energy flux: E(-AH) where H is enthalpy, produced by the isometric beating heart. 3. The value of ~t = [mechanical power]/[E(-AH)] is a measurement of myocardial efficiency; it varies from 0"08 to 0.26 in haemolymph or Ringer saline. 4. Efficiency is not affected by lowering PO2 in haemolymph but is modified by increasing K ÷ o r C a 2+ in isotonic salines. INTRODUCTION
(1954) that the hydrodynamic measurements are necessary. Thus, we were able to get closer to the actual work of the organ by using volumetric measurements on the ventricle perfused with hemolymph (Herold, 1966). However, the contraction activity of the heart only represents a part of the total energy brought into play by the myoeard. Only physical measurements of the quantity of heat released at the time of contraction can, under precise conditions, give an evaluation of the results of the energetic processes of the biological system i.e. Y_,(-AH) where H represents enthalpy. However, the technical problems of measuring the heat production of the beating heart are even more complex than when using ergometric measurements. The linear thermo couples developed by Hill (1931) on the skeletal muscle can not be applied to the heart which contracts in three spatial dimensions. There, once again, the answer is to use papillary muscles or cardiac strips which can be assimilated to linear elements and then investigated using the thermo-couples technique (Ricchiuti & Gibbs 1965; Grayson et al., 1971). However, the microcalorimeter techniques developed by Calvet & Prat (1963) are, as Boivinet (1971) pointed out, perfectly suited to the study of biological phenomena having a low production of heat over a long period. Thus, instead of trying to make an approximation of the thermic process of a contraction it is possible to explore the variations in thermic rate of the heart over a period of many hours and thus obtain the integration of energetic production of the myocard. This microcalorimeter technique, developed by Boivinet & Rybak (1969) on a frog heart and by Herold & Cudey (1972) on a snail heart, has shown that the good reproducibility of the thermograms can give probable correlations between the mechanical activity and heat production which take into account the total energetic production of the myocard.
THE STUDY of the energetic characteristics of the isolated heart requires firstly a good knowledge of the mechanical work of the organ and secondly a reproducible measurement of the total energy flux of the isolated biological system. In both cases the difficulties are considerable due to the fact that the heart is a complex mechanism with a low survival rate. With both vertebrate and invertebrate animals the best approach to cardiac work depends on a prior choice of measuring techniques. Thus, Siess et al. (1970) when working on the isolated guinea pig, evaluated the work using the product of the force by displacement at known frequencies expressed in g/cm per hr. To do this they used a displacement transducer of the isotonic lever type. Burns et al. (1972) working on a dog heart, decided to measure the volume variations with a micro-manometer placed in the left ventricle and then connected to a ratemeter, This experiment enabled them to evaluate the isovolume and isotonic work in g/cm 2 per cm. However, if this technique takes into account the anatomical characteristics of the hollow heart muscle it can only be applied to one of these cavities. Due to these inherent difficulties some authors have further developed the Fenn (1923) technique for normal striated muscle and preferred to continue their research on cardiac strips or papillary muscle fragments assimilated with linear elements (Whalen 1961; Strauer, 1973). In the case of a heart with a more simple structure such as that of molluscs the problem is the same except that the survival of the organ is easier to maintain. This work can be measured with the mechanoelectric transducer R.C.A. 5734 which gives values for the force-displacement product in relation to frequency. This transducer was used by Paul (1961); Hill & Schunke (1967) and Almqvist (1973) on the Helix heart and gave an estimation of the dynamic properties of the organ. However, the linear displacement recorded under these conditions did not take into account the contraction of the transversal and oblique MATERIALS A N D METHODS fibers of the heart wall. The hearts came from an homogeneous batch of winter So as to take into consideration the true and full work of the heart we believe, like Schwartzkopff snails (Helix pomatia L.) that the laboratory received in 435
436
J . P . HEROLD
November. These snails were room at temperatures between was between 17-5 and 22 g and heart was 28.6 _ 1-9 rag. Their 79-84 to 83.18~o.
then kept in a darkened 7 and 12°C. Their weight the average weight of the water content varied from
I. Measurement o f ventricle work Part of the shell was taken off at the level of the heart. An incision was made in the pericardium, then another in the auricula so as to push the oval end of the straub canula (l mm diameter) back to the inside of the ventricle cavity. A ligature was made at the level of the auriculoventricle junction and the organ was fixed onto the canula. The aortic end was then ligatured. The section of the auricular strip and the aorta was then free. The isolated heart fixed on the canula became part of a volumetric system with constant pressure. One end of a polyethylene tube fitted perfectly onto the perfusion canula. The other end was linked to a horizontal semi-capillary tube graduated in microliters. A liquid index (coloured water with a lubricator added) moved in the tube at each ventricle systol. The volume measured between the index limits displaced at each cardiac revolution represented exactly what entered the ventricle. The advantage of this system is twofold: its great simplicity and its very low inertia. One variable which can be modified at will is available. This is the height of the perfusion liquid in the canula and this means that conditions of stable and known activity can be imposed. So as to remain within the physiological conditions this parameter can only be increased within reasonable proportions. When going from 2 to 8 cm the ergometric characteristics of the heart are considerably modified: above this there appears to be a dissociation of the contraction of the different muscular zones of the organ. To fulfill the requirements set out by Assendelft et al., 1973, who established the units of the International System (S.I.) recommended in Physiology, we will express the results in power units:watt. As well the advantage of using the International System means that the values of mechanical power can be compared to the total power measured by the microcalorimetric technique. The mechanical power of the isolated ventricle is calculated as follows: P = I.t.g.h.V.N
/~ =
g I1
=
N=
specific weight of the perfusion liquid in kg/m 3 i.e. for hemolymph: 1.013.103 acceleration of the weight 9.81 m/s: height of the perfusion liquid in the canula in m systolic volume read off in ttl. i.e. m 3 . 10 -9 frequency in systols per min. To remain in the physiological values and not to use fractions of systols we have kept the frequency in systols m n - ~ instead of expressing them in Hertz (systols per sec). This is the same as calculating the power in J. s-~ i.e.W.
P is then expressed in watts 10 -6. The measurements are taken 30 min to an hour after preparing the hearts when their automatic activity is stable and regular. 2. Measurement o f ventricle heat production A complete study of the Calvet microcalorimeter is available in "Recent progress in microcalorimetry" Calvet & Prat (1963) and in "Biochemical microcalorimetry" Brown (I 969). The electrical value is measured by the electromotive force (e.m.f.) of the thermo-piles which is proportional to the energy flux between two units. The e.m.f, is measured by the deviation A of a galvanometer which at each moment is proportional to the flux. We have insisted on the use of a differential apparatus (Herold & Cudey, 1972) so as to obtain reproducible ther-
mograms. It is the association in opposition of two microcalorimetric elements having the outer cell at the same temperature 0~,. If 0~,.~ is the temperature of the inside cell of the first element, the thermocouples supply an e.m.f.: ER = n Z(01,.i - 0~) the e.m.f, corresponds to the second element at a temperature of 0in.2 E 2 .= n Y-(0in.2 - 0,.~) the results of the two opposing piles is E = E I - E2
= n E ( 0 i n . i - - 01n.2 )
This procedure makes it possible to eliminate by compensation the parasite phenomena disturbing the measurements on the condition that the two microcalorimetric cells receive the same energy at the same time. There is an advantage: if the external temperature variations are the same for the two cells the e.m.f, result is independent of these variations and the stability of the experimental zero is assured. This was the case in our experiments when the two ceils received an equal and constant flow of gas which assured a stable P O , which was necessary for the survival of the hearts. The calorific power developed by the biological material in the measurement cell was then obtained by the basic Tian equation: w=_PA g where P is the leakage coefficient of the microcalorimetric element in watts per degree. g is the sensitivity constant of the galvanometer in meters per degree. The equation P/g is thus a power divided by a length and it is expressed in watts per meter. It also defines the sensitivity of the calorimeter. Our apparatus had a Pig = 4.90.10 .3 w/m. The experimental accuracy is of the order of 0-8~ The detection threshold in watts is 0.2.10-6 watt i.e. a fifth of a microwatt. A is the deviation of the galvanometer recorder. Thus, in our experiment, W is a power expressed in watt 10 -6" The laboratory cell which takes the ventricles has a volume of 10ml. It was thus not possible to perfuse one or more ventricles. To be sure of having a sufficient heat flow and to obtain accuracy we used at one time l0 ventricles functioning isometrically and stretched on a metal frame. A tension of between 150 and 650 mg was selected at the beginning of the experiment. The frame with the hearts was put into hemolymph or physiological saline which was maintained at a steady PO_, with a gas bubbling (mixture O2-N2) or air P O , = 150 Torr (40 ml/hr per cell). The control cell contained the same volume of saline and received the same gas flow. The opposing cells thus eliminated the parasitic effects due to the expansion of gas or to the vaporization of the liquid in the cells. The reading of A was taken after the initial thermic stabilization i.e. I hr after the cells had been put in the calorimeter. The base reference line or the experimental zero was obtained at the end of the experiment with the injection of a 10~o formol solution which eliminated all thermogenesis.
RESULTS T h e contractile activity o f the heart, its m e c h a n i c a l p o w e r d e v e l o p e d a g a i n s t a n i m p o s e d pressure, only r e p r e s e n t a p a r t o f the total activity that is b r o u g h t into play by the m y o c a r d . O t h e r energetic a s p e c t s were linked to m e t a b o l i c
Myocardial efficiency in isolated ventricle of the snail exchanges such as phosphorylations, decarboxylations, deaminations, and synthesis phenomena. The energy linked to the maintenance of the ionic gradient with its bioelectric consequences also played a part. What is interesting about the microcalorimeter is that it measures the result of all these processes without taking into consideration the intermediary metabolism. At one moment t the deviation of the galvanometer recorder represented the total energy developed by the biological system i.e. E ( - A H ) where H is the enthalpy. It was thus possible to compare the values of the mechanical power with the total power, this relationship represented the efficiency of the biological system. We adopted the same efficient relation as Gibbs et al., 1967: mechanical power 0~.~-
437 /
1 / l
/
PO2 "15 torr : y ,0-937 x + 6.536
J
/" ~ hernolyml~
f
/
Cag÷:y • 1x+8.720
/
./
//"
4~ PO:z,fSO tort : y • 0-755 x+ 6. 213 /~inger : y-0"780 x+ 6 - 0 3 8
,oh/
5
Z(-AH)
1. Determination of the efficiency of the active heart in hemolymph (a) Mechanical power. When the ventricle was perfused with hemolymph at PO2 constant at 150 Torr, the work developed by the organ increased in relation to the height h of the liquid in the perfusion canula. The statistical analysis of the results showed that there is a positive linear correlation (r = +0'992) between the volume of the ventricle systolic volume V fy = 8.9) and the mechanical power P (y = 3.56). It can be represented: y = 0-755 Z + 6"213 (b) Total power. The total energy developed by the heart and measured with the Calvet microcalorimeter at PO_, = 150 Torr was obtained on isometric beating organs submitted to tensions that increase from 150 to 650 rag. It must be mentioned that as a non-stretched heart does not give any mechanical power it gives an energy of 5-8 ___ 1-2.10 - 6 watt which corresponds to the rest metabolism of the organ. (c) Calculation of the efficiency coefficient of ~t. The accuracy of results of the two types of measurements could be considered to be satisfactory, but the expression of ~ remained approximate because the experimental conditions are different. A critical examination shows: (a) the mechanical power measurement was done on hearts perfused with hemolymph at variable pressure (p.g.h.)
I
5 P,
I0 w a t t I 0 "e
Fig. I. Linear correlation between the ventricle systolic volume V, and the mechanical power P of the isolated ventricle perfused with hemolymph (at two values for PO2) and with isotonic saline solutions, equilibrated Ringer solution and enriched with K + or Ca 2+. (b) The total power measurement was done on hearts under variable tensions The pressure-tension correspondance must be adjusted beforehand from a known correlation between the systol frequency and the pressure or tension. Thus, a pressure corresponding to h = 2.10 -2 or a tension of 150 mg imposes a frequency of 32 _ 4 systols m n - t on the heart; whereas for h = 8.10 -2 or 650 mg the frequency is 53 + 5 systois m n - t. The comparison of ergometric and microcalorimetric results is thus possible, but only gives the expression of an approximate efficiency. In the case of an active isolated heart in contact with hemolymph the efficiency relation varies between 0"08 and 0"26. These results can be compared with those obtained by Bing & Michal (1959) using a rat heart where ct = 0.20-0.25 and by Reissman & van Citers, 1959 using the same preparation where ct = 0.07-0.30 according to the frequency of the heart.
2. Factors having an effect on efficiency (a) PO2 of the biological saline. During previous research we have shown that the PO2 of the hemolymph is a factor limiting the heat production of the
Table 1. Mechanical power of an isolated ventricle perfused with hemolymph. Results with standard error, and number of experiments ( ) h.m
10 -2
V.m a . 10 -9
N. systoles mn- i P.watt l0 -6
2__.0"1 6"8 _+ 0'5 34 -+ 3 0.76 _ 0.21 (12)
2___0"1 7.3 -I- 0"4 37 _+ 4 1.78 _ 0.18 (12)
6_+0"1 9'6 -+ 0"5 42 _+ 4 3.99 -+ 0.15 (12)
8_+0'1 11'9 -+ 0'6 49 _+ 5 7.71 _+ 0'16 (11)
Table 2. Total power of the active isolated ventricle in hemolymph Tension mg Z(-AH) watt. l0 -~
150 9.3 4- 0'82 (6)
300 16'5 -I- 1-4 (6)
500 19.2 _ 2'3 (5)
650 29-9 _ 3.4 (5)
438
J.P. HEROLD
I0-
L~ O x 13 4-
Hemolymph P02,15tocr HemOlymph POz-150~rr Rin lit
x
CO~ K~
÷
.'o_ O
x+
5-[3
0
&,
x+
a 04+
~
o
oI
I
IO
I
3O
20
Z (-L~H),
I 40
wott I0 -6
Fig. 2. Graph of the values of the efficiency relations ct
mechanical power = P total power: Z( - AH)
or-Equilibrated physiological saline of the Ringer type. This solution contains in m M / l : NaCI: 73. KCI:
of the ventricle isolated under different conditions: in contact with either hemolymph or isotonic saline solutions. Table 3. Mechanical power and total power of a ventricle isolated at PO2 = 150 Torr P watt 10-* E ( - A H ) watt x
10 - 6
0-76 9-3 0'08
1'78 3.99 7.71 16.5 19.2 2 9 . 9 O"11 0.21 0.26
isolated heart (Herold & Cudey, 1972). Thus, if the enthalpy of the biological system is affected by the PO2 it can be thought that the efficiency relation also depends on it too. The mechanical power and the total power are measured at PO_, = 15 (2~ 02 in N2) maintained constant during the experiment. This value of PO_, was chosen for two reasons: it corresponds to the physiological values in oivo in the plumonary vein of the intact snail; and it is close to the PO250 of the hemocyanine at 20°C i.e. 12 Torr (Redfield, 1934). Under these conditions the comparison of the ergometric and the calorimetric results give the following efficiency values: Table 4. Mechanical power and total power of an isolated ventricle at PO2 = 15 Torr in Hemolymph P watt 10 - 6 5"(-AH) watt 10-6 :t
0'51 6.1 0"08
(b) Composition of the physiological saline. To determine the influence of the ionic composition of the physiological saline on the energetic characteristics of the ventricle we used a Ringer solution as a reference solution as the mineral composition is very similar to that of hemolymph (Burton 1965; Meincke, 1972). Then to show the respective role of the K + and Ca 2 ÷ ions we made enriched solutions of each of these ions and maintained the isotony with NaC1 so that the heart would maintain its automatic properties. It is known that the K ÷ C a 2+ ions have marked antagonistic effects on the mechanical activity of the isolated heart (Jullien, 1936). The problem is to determine if the cardiac efficiency is changed when the ionic relationship is experimentally modified.
1.39 3'29 6.19 12.3 1 4 . 6 21.7 0'11 0"23 0"28
It was found that the work and the total power are lower than those given by the active ventricle at 150 Torr. However, the efficiency relation is hardly different from that obtained under much better oxygenation conditions. This clearly shows that the myocard keeps a metabolism intact with very low oxygenation rates done in vitro.
4. CaCI2: 10.8. pH = 7.2-7-6 and maintains the automatism of the ventricle for many hours. When perfused with this solution the isolated ventricle has the following mechanical performances (Table 5). In this case a positive linear correlation was found between the volume of ventricle systolic volume and the mechanical power. The correlation was y = 0'780Z + 6-038 (r = 0'998) and showed that in this equilibrated solution the mechanical characteristics of the ventricle were very similar to those measured in hemolymph, Compared with the total power obtained using the microcalorimetric technique the efficiency relationship varies in relation to the strain that the ventricle undergoes within the same limits as those defined in hemolymph. Table 6. Mechanical power and total power of an isolated ventricle in ringers solution (PO2 = 150 Torr) P watt 10-6 5"(-AH) watt 10-~'
0.88 2.68 5.7 11-06 12.7 1 7 . 4 2 5 - 4 41.76 0.06 0.15 0'22 0.26
fl-Calcium enriched solution without potassium. A concentrated solution of CaCI2 = 15. NaCl = 72.8 mM/1 maintained the automatic activity of the isolated heart. The mechanogram gave a reduced frequency of 4 0 ~ in relation to the Ringer solution accompanied by a reduced diastolic tonus. However, the amplitude of the systols was increased ( + 2 5 ~ ) . The results coincide with those of Paul (1961) which underlined the fundamental difference of the Helix heart response to calcium in relation to the response of the vertebrate heart where an excess of this ion brings about a systolic arrest. The linear correlation between the volume of the systolic volume and the mechanical power gave y = l Z + 8.720 (r = +0.988). Compared with the total power the mechanical power modified the efficiency relation.
Table 5. Mechanical power of the isolated ventricle perfused with ringer solution (PO2 = 150 Tort) h.m 10-2 V . m 3 10 - 9
N.systoles.mn -t P.watt 10-6
2___0.1 6'6 _ 0-4 41 _ 3 0.88 _ 0'18 (9)
4+0.1 8"2 -I- 0"3 50 _ 4 2-68 + 0.16 (10)
6-1-0.1 10"6 _ 0"4 55 _ 4 5-7 _ 0.16 (10)
8-1-0.1 14'6 4- 0'5 58 _ 4 11.06 -t- 0-14 (10)
Myocardial efficiency in isolated ventricle of the snail Table 7. Efficiencyof the ventricle in a solution enriched with Ca -'+ (15 mM CaCI2, 72-8 mM NaCI) P watt 10-6 Z(-AH) watt 10-6 ~t
0'56 1.53 3.48 5.75 4.6 5'2 9'3 16.6 0'12 0.29 0.37 0'34
The ventricle performance is thus increased with the presence of Ca -'+ although the work and total power values were respectively decreased by an excess of Ca-"+. This phenomena, which was accompanied by a hyperpolarization of the muscle fiber (Burton & Loudon, 1972)was however accompanied by a better efficiency. ~-Potassium enriched solution without calcium. The solution used contained KCI = 8, NaCI = 79-7 mM/l. A mechanical activity of weak amplitude was maintained (-30%), but the frequency was high (+25%). The diastolic tonus was increased (+ 40%) in relation to the Ringer solution. The linear correlation between the volume of the ventricle and the mechanical power gave y = 0.843;( + 4-319 (r = +0.994). The ventricle efficiency was reduced in relation to the balanced solution. Table 8. Efficiencyof the ventricle in a solution of 8 mM KCI and 79.7 NaCI P watt 10-* E(-AH) watt 10-* ~t
0'57 2 ' 1 8 5 ' 3 2 8'57 6.9 19'2 2 6 ' 8 44.8 0'08 fill 0 ' 1 9 0"19
In this case the total power is higher than that measured in the equilibrated solution, whereas the work was considerably decreased. The could be explained by the onset of a cellular alteration brought about by the solution. Elekes et al. (1973) have shown that ultrastructural alterations of the sarcotubular cisternae in the myocardial cells incubated in a solution without Ca 2÷ take place. However, if the efficiency was low, the total power increase in relation to the equilibrated solution suggests that the overall metabolisms were activated. This hypothesis would seem to be confirmed by the fact that under these same conditions the myocardial respiratory exchanges were increased (Herold, 1966) and the excitability of the tissues was increased as the depolarization of the myocardial fibers would prove (Burton & Loudon, 1972).
439
The myocardial efficiency relation can be estab-. lished when it is submitted to different strain conditions. The results show that the efficiency can vary from 0'06 to 0'26 when the ventricle is in contact with hemolymph or a well balanced mineral solution. A decrease of PO-' in the hemolymph does not modify the efficiency although the work and total power are both affected. However, a modification of the ion balance brings about efficiency variations. The K ÷ and Ca -'+ ions have antagonistic effects if both reduce the mechanical power, the enthalpy variation is increased by K ÷ and reduced by Ca 2÷. The measurement of ~ ( - AH) can give an estimation of the tissue excitability. SUMMARY
Work of isolated ventricle of a Mollusc is measured by using perfusion techniques. It is possible to measure the total mechanical power due to all muscular element activity, whatever their spatial arrangement is. The Calvet's microcalorimeter gives, with a good approximation, the total energy flux (~(-AH), where H is enthalpy) resulting from the different energetic aspects: isometric work and metabolism of the biological system. The technical difficulty is to obtain reproducible thermograms when ventricles are in standard conditions of tension and oxygenation. This is possible by means of differential records in two opposite microcalorimetric elements. Then, myocardial efficiency can be obtained under different tensions. Results show that efficiency increases with strain from 0"06 to 0'26, whatever the medium is: hemolymph or equilibrated physiological saline. Lowering the PO2 in hemolymph does not affect myocardial efficiency, although work and total energy are affected. When the ionic balance is altered, efficiency becomes modified: K + and Ca -'+ have antagonistic effects, the two ions lower the mechanical power, but total energy flux is increased by K + and lowered by Ca -'+. Measurements of E ( - A H ) can estimate myocardial excitability. Acknowledgements--I would like to express my thanks to Professor J. Ripplinger who started me off on this work. My thanks also go to G. Cudey whose help with the microcalorimeter was invaluable and to M. Nicolet who looked after the statistical analysis.
CONCLUSION In an isolated Mollusc ventricle the work was measured on the perfused organ and this way it was possible to measure the mechanical power resulting from the activity of all the muscular elements whatever their spatial arrangement were. The Calvet microcalorimeter gave a good approximation for the enthalpy variation resulting from the different energetic aspects, isometric work and metabolic exchanges of the biological system. The technical difficulty was to obtain reproducible thermograms when the ventricles were placed in precise conditions of tension and oxygenation. This is possible due to the differential recordings from the two opposite microcalorimetric elements.
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BOIVINETP. & RYaAK B. (1964) Etude microcalorim6trique du fonctionnement 61ectrom6canique du coeur de grenouille. L/fe Sci. 8, part. If, l 1-20. BURNS J. W. & COVELL J. W. (1972) Myocardial oxygen consumption during isotonic and isovolumic contractions in the intact heart. Am. J. Physiol. 223, (6) 14911496. BURTON R. F. & LOUDON J. R. (1972) The antagonistic actions of calcium and magnesium on the superfused ventricle of the snail Helix pomatia. J. Physiol., Lond. 220, 363-381. CALVET E. '~" PRAT H. (1963) Recent progress in Microcalorimetry. Academic Press. New York. ELEKES K., KISS T. & KATALIN S-ROZSA. (1973) Effect of Ca-free medium on the ultrastructure and excitability of the myocardial cells of the snail, Helix pomatia L. J. molec. Cell. Cardiol. 5, 133-138. GIBaS C. L., MOMMAERTS W. F. H. M. & RICCHIUTI N. V. (1967) Energetics of cardiac contractions, d. Physiol., Lond. 191. 25--46. GRAVSON J., COULSONR. L. & WINCHESTER B. (1971) Internal calorimetry, assessment of myocardial blood flow and heart production. J. appl. Physiol. 30, (2) 251-256. HEROLD J. P. (1966) Consommation d'oxyg6ne et travail du coeur isol6 d'Escargot Helix pomatia L. C.r. Soc. Biol. 160, (7) 1442-1445. HEROLD J. P. & CUDEV G. (1972) La PO2: facteur limitant de la thermog6n6se du coeur de I'escargot. J. Physiol., Paris, 65, (3) 423 A. HEROLD J. P. & ConEY G. (1972) Approche de 1'6nerg6tique cardiaque d'un Mollusque par la microcalorim6trie. C.r. Soc. Biol. 166, (4/5) 561-564. HILL A. V. (1931) Myothermie experiments on the frog's gastrocnemius. Proc. R. Soc. B. 109, 267-303. HILL R. B. & SCHUNKE P. J. (1967) Contractility cycle of an isolated gastropod ventricle. Experientia 23, 570, 1-6.
JULLIEN A. (1936) Des r6actions compar~es des coeurs de Vert6br6s et d'Invert6br6s vis h vis des ~lectrolytes et des drogues. Th~se Bailliere J. B., Paris. MEXNCKE K. F. 0972) Osmotischer Druck und ionale Zusammensetzung des Hiimolymphe winterschlafender Helix pomatia bei Konstanter und sich zyklisch iindernder Temperatur. Z. vergl. Physiol. 76. 226-232. PAUL O. H. (1961) Effects of calcium on the spontaneous contractions of the isolated ventricle of the snail Helix pomatia. Experientia, 17, (7) 310-311. REDFIELD A. C. (1934) The Haemocyanins. Biol. Reos. Cambridge. Phil. Soc. 9, 175-212. REISSMAN K. R. & VAN CITERS R. L. (1959) Oxygen consumption and mechanical efficiency of the hypothermic heart. J. appl. Physiol. 9, 427-430. RICCmUTI N. V. & Gmas C. L. (1965) Heat production in a cardiac contraction. Nature N.Y. 5013. 897-898. SCHWARTZKOPFF J. (1954) Uber die Leistung des isolierten Hertzens der Weinbergschnecke (Helix pomatia L.) im Kiinstlichen Kreislauf. Z. vergl. Physiol. 36, 543-594. SIESS M., KELLER H. J., SCHARE E. & GEISSLER J. (1970) The continuous and simultaneous measurement of 0 2consumption, rate of decarboxylation of ~4C-substrates and the performance of spontaneously-beating isolated heart atria of Guinea pigs. J. molee. Cell. Cardiol. 1, 261-269. STRAUER B. E. (1973) Force-velocity relations of isotonic relaxation in mammalian heart muscle. Am. J. Physiol. 224, (2) 431-434. WHALEN W. J. (1961) The relation of work and oxygen consumption in isolated strips of cat and rat myocardium. J. Physiol., Lond. 157. 1-17.
Key Word Index--Helix pomatia; isolated ventricle; perfusion; mechanical power; Calvet microcalorimetry; enthalpy; efficiency.
