27, 539-546 (I%@)
CRYOBIOLOGY
Effects of Low Temperature on Contraction in Papillary Muscles from Rabbit, Rat, and Hedgehog BIN LIU,’ BJORN WOHLFART, AND BENGT W. JOHANSSON Department
of Pharmacology,
University of Lund, 223 62 Lund and Heart Section, General Hospital, 214 01 Mulmii, Sweden
During hibernation the body temperature may fall to only a few degrees above 0°C. The heart of the hedgehog continues to function whereas the hearts of nonhibernating mammals stop beating. The present study was performed to investigate and compare the mechanical responses to hypothermia in rabbits, rats, and hedgehogs. Isometric force was recorded from papillary muscles mounted in an organ bath and effects of hypothermia on the mechanical restitution curve were also compared. A reduction of bath temperature from 35°C caused an increase in peak developed force. Maximum force was seen at 20°C in the rabbit, 15°C in the rat, and 10°Cin the hedgehog preparations. In all the species there was a similar prolongation of time to peak force and of time from peak to half-relaxation as temperature was lowered. An increase in resting force and after-contractions were recorded in the rabbit and rat muscles at temperatures below 15 and lO*C, respectively. The rabbit and rat preparations became inexcitable at temperatures below IO and 5°C respectively. The hedgehog papillary muscle, on the other hand, still contracted at 0°C and did not show increased resting force nor after-contractions. The results are consistent with the hypothesis that there is a calcium overload in cardiac cells from rabbit and rat at low temperatures but there is no calcium overload in the hedgehog muscle during hypothermia. o 1980Academic FESS, inc.
Lowering of temperature has profound effects on the heart. Many mammals, including the human being, develop ventricular fibrillation or other cardiac arrhythmias during hypothermia (4, 23, 24, 43). The isolated heart from nonhibernating mammals usually ceases to contract at temperatures slightly above 10°C. The hedgehog is a hibernator, and thus has the capacity to decrease its body temperature to a few degrees above 0°C. The isolated heart of hibernators continues to contract at about 0°C (7, 20, 38) and transmembrane potentials around - 50 mV can be recorded at low temperatures (13, 21, 36, 39, 48). In
Received June 22, 1988; accepted November 27, 1989. r Permanent address: Department of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G ZE9.
the present study we have compared the mechanical responses of papillary muscles from hedgehogs with those of rabbits and rats at low temperatures. METHODS
The experimental procedures were similar to those described in our previous report (37). Rabbits (35-42 days old) and rats (about 42 days old) were killed by cervical dislocation. The experiments on summer active hedgehogs were performed in June. The hedgehogs (Erinaceus europaeus, 70& 1300 g) were anesthetized with thiopenthal sodium (30 mg/kg). The right papillary muscles were excised and mounted horizontally in a thermostatically controlled chamber. The muscle length was 95% of that for maximal force production. The Tyrode solution was continuously perfused using a recircu-
539 001l-224OBo $3.00 Copyhht 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
540
LIU,
WOHLFART,
lation system, A digital thermistor probe monitored the bath temperature close to the muscle. The preparations were equilibrated at 35°C at least 60 min before the recordings. In a transient change of temperature experiment, the temperature of the chamber was decreased continuously from 35 to 0°C at a rate of 0.7”C/min and then rewarming took place. In the steady state experiments the temperature was held at 25, 20, 15, IO, 5, and 0°C for at least a period of 30 min for equilibration. The muscle was stimulated at a frequency of 30/min through a pair of platinum wire electrodes. At bath temperatures below 15°Ca frequency of 12/ min was used. In the mechanical restitution experiments the same stimulus protocol as in the accompanying study (37) was used. Isometric force was monitored on an oscilloscope and recorded on a Grass Polygraph ink-writer. The signal was also fed into a Luxor ABC 800 computer (1000 Hz sampling rate) for analysesof (1) peak force (F), (2) maximal rate of force development (dF/ dt max), (3) time from stimulus to peak force (TPF), (4) time from peak force to half relaxation (THR). RESULTS
Transient Eflecfs of Lowering the Bath Temperature
The temperature was continuously decreased from 35 to 0°C at a rate of O.TC/min. During the course of lowering temperature, the papillary muscles were paced at 1Ymin. In rabbit papillary muscle, the amplitude of the contraction increased with lowering of temperature and reached a maximum at about 20 to 15°C. Below 15”C, the amplitude of contraction declined but the resting tension increased markedly. When temperature was decreased further, after-contractions were seen and the muscle preparation did not respond to every stimulus pulse. An increment of resting tension and after-contractions were also ob-
AND
JOHANSSON
served in the rat papillary muscle at low temperatures. On lowering temperature in the hedgehog papillary muscle, however, the resting tension actually decreased somewhat, no after-contractions were observed, and the muscle maintained regular contractions even at temperatures close to 0°C. The effects of rewarming the muscle preparations from about 0°C is demonstrated in Fig. 1. Isometric contractions were continuously recorded with a slow paper speed as temperature was gradually increased toward 30 “C. The effects on resting force, contraction amplitude, and aftercontractions, as described above, were all reversible on rewarming the preparations. Constant Temperature Experiments
In the constant temperature experiments, the papillary muscles were equilibrated at each temperature for at least 30 min. Four parameters of force production were analyzed as a function of temperature and are described below. Figure 2A shows peak force of the three speciesrelated to the temperature. In papillary musclesfrom rabbits, peak force increased about five times as temperature was decreased from 35 to 20°C. At lower temperatures there was a reduction in force and the contraction disappeared at temperatures below about 10°C.In rat papillary muscles, the maximal value of peak force was seen at about 15°C and the preparations became inexcitable below 5°C. In hedgehog papillary muscles, the maximum of peak force was recorded at 10°C and the contraction was still maintained at 0°C. As can be seen in Fig. 2A, peak force in rat and hedgehog muscles did not change so much with temperature as did the rabbit ventricular muscle. Figure 28 shows the maximal rate of force production (dF/dt max) related to temperature in the three species. It can be seen in the figure that dF/dt max increased
HYPOTHERMIC
EFFECTS ON PAPILLARY
541
MUSCLES
Rabbit
t 5’c
t 15-c
t 1O’c
t 25°C
t 20-c
t 3Ok
FIG. 1. Continuous paper recordings of isometric contractions (pacing frequency lZ/min) of papillary muscles from rabbit, rat, and hedgehog. The preparations were initially held at 35°C and the temperature was decreased toward 0°C. The figure shows the effects of rewarming.
in rabbit papillary muscle when temperature was reduced from 35 to 15°C and dF/dt max decreased markedly between 15 and 10°C. However, in rat and hedgehog papillary muscles dF/dt max decreased with reduction of temperature from 35°C. Below about 10°C dF/dt max did not change significantly in the rat and hedgehog papillary muscles as is clear from the figure. The effects of temperature on time from
stimulus to peak force (TPF) are demonstrated in Fig. 3A. The prolongation of TPF with lowering of temperature was very similar in the rabbit, rat, and hedgehog preparations. (Values are expressed as percentages of TPF at 35°C in the three species, respectively.) Time from peak force to halfrelaxation (THR) was also analyzed as a function of temperature. With lowering the temperature, the relaxation phase was pro-
A
6001
T
Rat 1
35
I
I
30
25
1
20
1
’
15
10
Temperature
(‘c)
5
1
I
I
I
1
1
1
1
0
35
30
25
20
15
IO
5
Temperature
(‘c)
FIG, 2. (A) Peak force (+SEM) as a function of temperature in rabbit (n = 5), rat (n = 9), and hedgehog (n = 5) papillary muscles. (B) Effect of temperature on maximal rate of force production (&SEM) in the three species. Values expressed in percentage of steady state values at 37°C.
0
542
LIU, WOHLFART,
AND JOHANSSON
A 25001
2000 I
Hedgehog
1
3
1200-
FE F 60&
1
II
35
30
25
I
I,
20
15
Temperature
IJ
10
5
I
0
35
30
1
I
I
I
25
20
15
10
Temperature
(4~)
J
5
0
(‘c)
FIG. 3. (A) Time from stimulus to peak contractile force (?SEM) at different temperatures in rabbit, rat, and hedgehog papillary muscles. (B) Time from peak force to half-relaxation (THR) related to temperature. Values expressed in percentage of steady state values at 37°C. Same experiments as in Fig. 2.
longed as can be seen in the Fig. 3B. The below 10°C as can be seen in Fig, 4 (at the percentage of prolongation was about the same time there was also an increased resting force). In contrast, there were no aftersame in the three different preparations. After-contractions were regularly re- contractions in the hedgehog preparations, corded in rabbit papillary muscles at a tem- even at temperatures close to 0°C. Another sign of calcium overload at low perature of 13°C (Fig. 4). At the same time there was an increase in resting force as temperatures might be inferred from Fig, 5. described above (not shown in the figure). Mechanical restitution curves were reIn the rat papillary muscles, after-contrac- corded at 15°C in the three species, i.e., tions occurred as temperature was reduced peak force of a test contraction was related Rabbit
lOmN] h Rat
iomNl AL
A----
Hedgehog
lomNl A--IL A...- J-L..-13 “C
5 “C
0 “C
FIG. 4. Isometric contractions of rabbit, rat, and hedgehog papillary muschs at different temperatures as indicated in the figure. After-contractions are seen in the rabbit papillary muscle at 13°C and in the rat papillary muscle at 5°C. No after-contractions were recorded in the hedgehog preparations.
HYPOTHERMIC
EFFECTS
ON PAPILLARY
MUSCLES
543
was also a similar prolongation in all three species of time to peak force and of time from peak to half-relaxation as the temperature was lowered. An increase of peak isometric force of mammalian myocardium was also observed in other studies when the temperature was decreased to about 20°C (5,14,33,41,48). In skinned Purkinje fibres 01 , , , I of canine, the sensitivity of the myofila0.5 1.0 2.5 10 30 120 600 ments to calcium strongly increased when Test interval (s) temperature was lowered from 22 to 12°C FIG. 5. Mechanical restitution curves of rabbit, rat, (17) and time to peak force was markedly and hedgehog papillary muscles at a temperature of prolonged. This prolongation might be 15°C. The preparations were basically paced at a frecaused by a slowing of temperature-sensiquency of 0.5 Hz. At regular times a test pulse was introduced after a varied interval and peak force of the tive chemical reactions subsequent to a test contraction (FI max) was analyzed. The three me- very rapid calcium binding to Troponin C chanical restitution curves were nomalized to their (25). It would also follow, to some extent, maximal values (F,,J, respectively. from the temperature effects on calcium release and reuptake from the sarcoplasmic to the duration of the preceding test pulse reticulum (9,46). In a concomitant study on interval. This interval was varied from 0.5 rabbit, rat, and hedgehog cardiac preparato 600 sec. As the interval was prolonged, tions, we reported that the mechanical resforce increased in all three preparations. titution started later at low temperatures Beyond about 90 set there was a decline in (37). force in the hedgehog ventricular muscle. The regulation of intracellular calcium is However, there was no decline in force at important in cardiac cells (28, 29). Afterlonger intervals in the rabbit and rat mus- contractions are considered to be a sign of cles. calcium overload in the cell (2, 26,49), Any interference with the ability to maintain a DISCUSSION low intracellular calcium concentration In the present study we have compared would result in after-contractions and inthe cardiac mechanical restitution and con- creased resting tension (22, 30-32, 45). So far, two mechanisms are known to be tractions in hibernating (hedgehog) and nonhibernating (rabbit, rat) species. Adult related to calcium extrusion and mainteanimals of all three species were used, nance of low calcium concentration in carsince it has been shown that cold tolerance diac cells. The first is the Na-Ca exchange in which an entry of Na is coupled to the changes with development of individuals (1, 19). The data obtained showed that the transport of Ca from cytoplasmic to extracardiac muscle of hedgehog still contracted cellular space (15, 34, 42, 44, 45). The enat O”C, while the cardiac muscles of rabbit ergy for this exchange is derived from the and rat developed after-contractions and in- Na-K pump which actively transports Na creased resting force below 15 and lO*C, out of the cell and so establishes the Narespectively, and were not excitable below gradient across the membrane (8, 45). The 10 and 5°C respectively. A reduction in second is the ATP-dependent calcium temperature was also associated with an in- pump in myocardial sarcolemma (6, 12,47). crease in peak developed force. The opti- With radioactive 4SCa measurements, the mum was seen at 20°C in the rabbit, 15°C in calcium efflux was inhibited at low temperthe rat, and 10°C in the hedgehog. There ature in guinea pig myocardium (44). In re-
544
LIU, WOHLFART,
cent studies with fresh red cells from hibernating species (hedgehogand ground squirrel) there was an active calcium efflux when the cells were incubated at 5°C. However, fresh red cells from guinea pig, or ATP depleted red cells from hedgehogand ground squirrel, showed a significant accumulation of intracellular calcium at low temperatures (16, 18). In the present study the following signs of cellular calcium accumulation in response to hypothermia were observed: (1) Increased resting force, (2) after-contractions, and (3) a reduced decline of force after the maximum in the mechanical restitution curve at the longest test intervals (Fig. 5). (This probably reflects a reduced cellular calcium outtlow.) Signs of calcium overload ((1) and (2) above) were seen at higher temperatures in the rabbit ventricular muscle as compared to that of the rat. No sign of calcium overload was seen in the hedgehog preparation. A rise of intracellular calcium above the normal diastolic value would activate a transient inward current (3, 10, 27, 35, 40). This might cause afterdepolarizations which could initiate extrasystoles and start severe arrhythmias in the heart (11). The greater cold tolerance of the hedgehog heart and its resistance to arrhythmias might to some extent be explained by the calcium handling system in the hedgehog myocardium preventing calcium overload at low temperatures. Further studies on calcium regulation in cardiac cells would provide more evidence for this hypothesis. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (14P-7920, 14X-184, 14X-08664)Ernhold Lundstriims Stiftelse and from the Faculty of Medicine, University of Lund. The stay in Sweden for Dr. Bin Liu was supported by the Swedish National Association against Heart and Chest Diseases. REFERENCES 1. Adolph, E. F. Responses to hypothermia in sev-
AND JOHANSSON eral species of infant mammals. Amer. J. Physiol. 166, 75-91 (1951). 2. Allen, D. G., Eisner, D. A., Pirolo, J. S., and Smith, G. L. The relationship between intracellular calcium and contraction in calciumoverloaded ferret papillary muscles. J. Physiol. 364, 169-182 (1985). 3. Arlock, P., and Katzung, B. G. Effects of sodium substitutes on transient inward current and tension in guinea-pig and ferret papillary muscle. J. Physiol. 360, 105-120 (1985). 4. Bi(irck, G., and Johansson, B. W. Comparative studies on temperature effects upon electrocardiogram in some vertebrates. Actu Physiol. Scand. 34, 257-272 (1955). 5. Blinks, J. R., and Koch-Weser, J. Physical factors in the analysis of the actions of drugs on myocardial contractility. Pharmacol. Rev. 15, 531600 (1%3). 6. Caroni, P., and Carafoli, E. An ATP-dependent Ca-pumping system on dog heart sarcolemma. Nature (London) 283, 765-767 (1980). 7. Chao, I., and Yeh, C. J. Temperature and activity of the excised perfused heart of the hedgehog. Chin. J. Physiol. 18, 17-30 (1951). 8. Chapman, R. A., Coray, A., and McCiuigan, J. A. S. Sodium/calcium exchange in mammalian ventricular muscle: study with sodiumsensitive microelectrodes. J. Physiol. 343, 253276 (1983). 9. Chiesi, M. Temperature-dependency of the functional activities of dog cardiac sarcoplasmic reticulum: a comparison with sarcoplasmic reticulum from rabbit and lobster muscle. J. Mol. Cell. Cardiol. 11, 245-259 (1979). IO. Colquhoun, D., Neher, E., Reuter, H., and Stevens, C. S. Inward current channels activated by intracellular calcium in cultured heart cells. Nature (London) 294, 752-754 (i981). I11. Cranefield, P. F. Action potentials, afterpotentials, and arrhythmias. Circ. Res. 41, 415-423 (1977). t2. Dhalla, N. S. Significance of Ca ATPases in the control of calcium movements in heart sarcolemma. J. Mol. Cell. Cardiol. 12, suppl I, No. 35 (1980). 13. Duker, G., Sjdquist, P-O., Svensson, O., Wohlfart, B., and Johansson, B. W. Hypothermic effects on cardiac action potentials: Difference between a hibernator, hedgehog and a nonhibemator, guinea-pig. In “Living in the Cold: Physiological and Biochemical Adaptations” (Helter, H. C., Mussachia, X. J., and Wang, L. C. H. Eds.), pp. 565-572 Elsevier, New York. 14. Edman, K. A. P., Mattiazzi, A. R., and Nilsson, E. The influence of temperature on the force-
HYPOTHERMIC
EFFECTS ON PAPILLARY
velocity relationship in rabbit papillary muscle. Stand. 90, 750-756 (1974). Eisner, D. A., and Lederer, W. J. Na-Ca exchange: stochiometry and electrogenicity. Amer. 1. Physiul. 248, C189-202 (1985). Ellory, J. C., and Hall, A. C. Ca” transport in hibernator and non-hibernator species red cell at low temperature. J. Physiol. 345, 148P (1983). Fabiato, A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J. Gem Physiol. 85, 247-289 (1985). Hall, A. C., Wolowyk, M. W., Wang, L. C. H., and Ellory, J. C. The effects of temperature on Ca transport in red cells from a hibernator (Spermophilus richardsonii). J. Therm. Biol. 12, 61-63 (1987). Hubbard, K. W., Harris, B. W., and Hilton, F. K. The effects of temperature on isolated hearts from neonatal through postweanling rats. Comp. Bbchem. Physiol. 70A, 491495 (1981). Hudson, J. W. Thermal sensitivity of isolatedperfused hearts from the ground squirrels, Citellus tereticandus and litellus tridecemlineatus. 2. Vergl. Physiologie 71, 342-349 (1971). Jacobs, H. K., and South, F. E. Effects of temperature on cardiac transmembrane potentials in hibernation. Amer. J. Physial. 203, 403409 (1976). Jensen, R. A., and Katzung, B. G. Simultaneously recorded oscillations in membrane potential and isometric contractile force from cardiac muscle. Nature (London) 217, 961-963 (1968). Johansson, B. W., BiBrck, G., Haeger, K., and Sjiistrtim B. Electrocardiographic observations on patients operated upon in hypothermia. Acta. h4ed. Stand. 155, 257-269 (1956). Johansson, B. W. The effect of aconitine, adrenaline and procain, and changes in the ionic concentration in the production of ventricular fibrillation in a hibernator (hedgehog) and a nonhibernator (guinea-pig) at different temperatures. Cardiologiu (Base0 43, 158-169 (1%3). Johnson, J. D., Charlton, S. C., and Potter, J. D. A fluorescence stopped flow analysis of Ca exchange with troponin C. J. Biool. Chem. 254, 3497-3507 (1979). Kass, R. S., Lederer, W. J., Tsien, R. W., and Weingart, R. Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibers. J. Physiol. 281, 187-208 (1978). Kass, R. S., and Tsien, R. W. Fluctuations in Actor Physiol.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34. 35.
36.
37.
38.
39.
40.
41.
MUSCLES
545
membrane current driven by intracellular calcium in cardiac Purkinje fibers. Biophys. J. 38, 259-269 (1982). Katz, A. M., and Tada, M. The “stone heart”: a challenge to the biochemist. Amer. J. Cardiol. 29, 578-580 (1972). Katz, A. M., and Reuter, H. Cellular calcium and cardiac cell death. Amer. J. Cardiol. 44, 188190 (1979). Kaufmann, R., Fleckenstein, A., and Antoni, H. Ursachen und Ausldsungsbedingungen von Myokard-Kontraktionen ohne Reguliires Aktionspotential. Pfliigers Arch. ges. Physiol. 278, 435-446 (1963). Kort, A. A., Lakatta, E. G., Marban, E., Stern, M. D., and Wier, W. G. Fluctuations in intracellular calcium concentrations and their effect on tonic tension in canine cardiac Purkinje fibers. J. Physiul. 367, 291-308 (1985). Lakatta, E. G., and Lappe, D. L. Diastolic scattered light fluctuations, resting force and twitch force in mammalian cardiac muscle. J. Physiol. 315,369-394 (1981). Langer, G. A., and Brady, A. J. The effect of temperature upon contraction and ionic exchange in rabbit ventricular myocardium. Relation to control of active state. J. Gerr. Physiol. 52, 682713 (1968). Lauger, G. A. Sodium-calcium exchange in the heart. Annu. Rev. Physiol. 44, 43M49 (1982). Lederer, W. J., and Tsien, R. W. Transient inward current underlying arrhythmogenic effects of cardiotonic steriods in Purkinje fibers. J. Physiol. 263, 75-100 (1976). Liu, B., Zhao, M., and Chao, I. Effect of cold on transmembrane potentials in cardiac cells of the hedgehog. J. Therm. Biul. l&77-80 (1987). Liu, B., Wohlfart, B., and Johansson, B. W. Mechanical restitution at different temperatures in papillary muscles from rabbit, rat, and hedgehog. CryobioIogy 27, (1990) Lyman, C. P., and Blinks, D. C. The effects of temperature on the isolated hearts of closely related hibernators and non-hibernators. J. Cell. Camp. Physiol. 54, 53-fj3 (1959). Marshall, J. M., and Willis, J. S. The effects of temperature on membrane potentials in isolated atria of the ground-squirrel, Citellus tridecemlineatus. J. Physiol. 164, 676 (1962). Matsuda, H., Noma, A., Kurachi, Y., and Irisawa, H. Transient depolarization and spontaneous voltage fluctuations in isolated single cells from guinea-pig ventricles. Circ. Res. 51, 142-151 (1982). Mattiazzi, A. R., and Nilsson, E. The influence of
546
LIU,
WOHLFART,
temperature on the time course of the mechanical activity in rabbit papillary muscle. Acm Physiol.
scnnd. 97, 310-318 (1974).
42. Mullins, L. J. A mechanism for Na/Ca transport. J. Gen. Physiol. 70, 681-695 (1977). 43. Nordrehaug, J. E. Sustained ventricular tibrillation in deep accidental hypothermia. Brii. Med. J. 284, 867-868 (1982). 44. Reuter, H., and Seitz, N. The dependence of calcium eftlux from cardiac muscle on temperature and external ion composition. J. Physiol. 195, 451-470 (1968). 45. Sheu, S. S., and Fozzard, H. Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development. J. Gen. Physiol. 80, 325-351 (1982).
AND JOHANSSON
46. Suko J. The effect of temperature on Ca’+uptake and Ca activated ATP hydrolysis by cardiac sarcoplasmic reticulum. Experientiu 29, 396 398 (1973). 47. Sulhake, P. V., and St Louis, P. T. Passive and active calcium fluxes across plasma membranes. Progr. Biophys. Med. Mol. Bioi. 35, 135-195 (1980). 48. Svensson, 0. L. O., Wohlfart, B., and Johansson, B. W. Hypothermic effects on action potential and force production of hedgehog and guinea pig papillary muscles. Cryobiology 25, 445450 (1988). 49. Tsien, R. W., Kass, R. S., and Weingart, R. Cellular and subcelhalr mechanisms of cardiac pacemaker oscillations. 1. Exp. Viol. 81, 205215 (1979).
CRYOBIOLOGY
Effects of Low Temperature on Contraction in Papillary Muscles from Rabbit, Rat, and Hedgehog BIN LIU,’ BJORN WOHLFART, AND BENGT W. JOHANSSON Department
of Pharmacology,
University of Lund, 223 62 Lund and Heart Section, General Hospital, 214 01 Mulmii, Sweden
During hibernation the body temperature may fall to only a few degrees above 0°C. The heart of the hedgehog continues to function whereas the hearts of nonhibernating mammals stop beating. The present study was performed to investigate and compare the mechanical responses to hypothermia in rabbits, rats, and hedgehogs. Isometric force was recorded from papillary muscles mounted in an organ bath and effects of hypothermia on the mechanical restitution curve were also compared. A reduction of bath temperature from 35°C caused an increase in peak developed force. Maximum force was seen at 20°C in the rabbit, 15°C in the rat, and 10°Cin the hedgehog preparations. In all the species there was a similar prolongation of time to peak force and of time from peak to half-relaxation as temperature was lowered. An increase in resting force and after-contractions were recorded in the rabbit and rat muscles at temperatures below 15 and lO*C, respectively. The rabbit and rat preparations became inexcitable at temperatures below IO and 5°C respectively. The hedgehog papillary muscle, on the other hand, still contracted at 0°C and did not show increased resting force nor after-contractions. The results are consistent with the hypothesis that there is a calcium overload in cardiac cells from rabbit and rat at low temperatures but there is no calcium overload in the hedgehog muscle during hypothermia. o 1980Academic FESS, inc.
Lowering of temperature has profound effects on the heart. Many mammals, including the human being, develop ventricular fibrillation or other cardiac arrhythmias during hypothermia (4, 23, 24, 43). The isolated heart from nonhibernating mammals usually ceases to contract at temperatures slightly above 10°C. The hedgehog is a hibernator, and thus has the capacity to decrease its body temperature to a few degrees above 0°C. The isolated heart of hibernators continues to contract at about 0°C (7, 20, 38) and transmembrane potentials around - 50 mV can be recorded at low temperatures (13, 21, 36, 39, 48). In
Received June 22, 1988; accepted November 27, 1989. r Permanent address: Department of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G ZE9.
the present study we have compared the mechanical responses of papillary muscles from hedgehogs with those of rabbits and rats at low temperatures. METHODS
The experimental procedures were similar to those described in our previous report (37). Rabbits (35-42 days old) and rats (about 42 days old) were killed by cervical dislocation. The experiments on summer active hedgehogs were performed in June. The hedgehogs (Erinaceus europaeus, 70& 1300 g) were anesthetized with thiopenthal sodium (30 mg/kg). The right papillary muscles were excised and mounted horizontally in a thermostatically controlled chamber. The muscle length was 95% of that for maximal force production. The Tyrode solution was continuously perfused using a recircu-
539 001l-224OBo $3.00 Copyhht 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
540
LIU,
WOHLFART,
lation system, A digital thermistor probe monitored the bath temperature close to the muscle. The preparations were equilibrated at 35°C at least 60 min before the recordings. In a transient change of temperature experiment, the temperature of the chamber was decreased continuously from 35 to 0°C at a rate of 0.7”C/min and then rewarming took place. In the steady state experiments the temperature was held at 25, 20, 15, IO, 5, and 0°C for at least a period of 30 min for equilibration. The muscle was stimulated at a frequency of 30/min through a pair of platinum wire electrodes. At bath temperatures below 15°Ca frequency of 12/ min was used. In the mechanical restitution experiments the same stimulus protocol as in the accompanying study (37) was used. Isometric force was monitored on an oscilloscope and recorded on a Grass Polygraph ink-writer. The signal was also fed into a Luxor ABC 800 computer (1000 Hz sampling rate) for analysesof (1) peak force (F), (2) maximal rate of force development (dF/ dt max), (3) time from stimulus to peak force (TPF), (4) time from peak force to half relaxation (THR). RESULTS
Transient Eflecfs of Lowering the Bath Temperature
The temperature was continuously decreased from 35 to 0°C at a rate of O.TC/min. During the course of lowering temperature, the papillary muscles were paced at 1Ymin. In rabbit papillary muscle, the amplitude of the contraction increased with lowering of temperature and reached a maximum at about 20 to 15°C. Below 15”C, the amplitude of contraction declined but the resting tension increased markedly. When temperature was decreased further, after-contractions were seen and the muscle preparation did not respond to every stimulus pulse. An increment of resting tension and after-contractions were also ob-
AND
JOHANSSON
served in the rat papillary muscle at low temperatures. On lowering temperature in the hedgehog papillary muscle, however, the resting tension actually decreased somewhat, no after-contractions were observed, and the muscle maintained regular contractions even at temperatures close to 0°C. The effects of rewarming the muscle preparations from about 0°C is demonstrated in Fig. 1. Isometric contractions were continuously recorded with a slow paper speed as temperature was gradually increased toward 30 “C. The effects on resting force, contraction amplitude, and aftercontractions, as described above, were all reversible on rewarming the preparations. Constant Temperature Experiments
In the constant temperature experiments, the papillary muscles were equilibrated at each temperature for at least 30 min. Four parameters of force production were analyzed as a function of temperature and are described below. Figure 2A shows peak force of the three speciesrelated to the temperature. In papillary musclesfrom rabbits, peak force increased about five times as temperature was decreased from 35 to 20°C. At lower temperatures there was a reduction in force and the contraction disappeared at temperatures below about 10°C.In rat papillary muscles, the maximal value of peak force was seen at about 15°C and the preparations became inexcitable below 5°C. In hedgehog papillary muscles, the maximum of peak force was recorded at 10°C and the contraction was still maintained at 0°C. As can be seen in Fig. 2A, peak force in rat and hedgehog muscles did not change so much with temperature as did the rabbit ventricular muscle. Figure 28 shows the maximal rate of force production (dF/dt max) related to temperature in the three species. It can be seen in the figure that dF/dt max increased
HYPOTHERMIC
EFFECTS ON PAPILLARY
541
MUSCLES
Rabbit
t 5’c
t 15-c
t 1O’c
t 25°C
t 20-c
t 3Ok
FIG. 1. Continuous paper recordings of isometric contractions (pacing frequency lZ/min) of papillary muscles from rabbit, rat, and hedgehog. The preparations were initially held at 35°C and the temperature was decreased toward 0°C. The figure shows the effects of rewarming.
in rabbit papillary muscle when temperature was reduced from 35 to 15°C and dF/dt max decreased markedly between 15 and 10°C. However, in rat and hedgehog papillary muscles dF/dt max decreased with reduction of temperature from 35°C. Below about 10°C dF/dt max did not change significantly in the rat and hedgehog papillary muscles as is clear from the figure. The effects of temperature on time from
stimulus to peak force (TPF) are demonstrated in Fig. 3A. The prolongation of TPF with lowering of temperature was very similar in the rabbit, rat, and hedgehog preparations. (Values are expressed as percentages of TPF at 35°C in the three species, respectively.) Time from peak force to halfrelaxation (THR) was also analyzed as a function of temperature. With lowering the temperature, the relaxation phase was pro-
A
6001
T
Rat 1
35
I
I
30
25
1
20
1
’
15
10
Temperature
(‘c)
5
1
I
I
I
1
1
1
1
0
35
30
25
20
15
IO
5
Temperature
(‘c)
FIG, 2. (A) Peak force (+SEM) as a function of temperature in rabbit (n = 5), rat (n = 9), and hedgehog (n = 5) papillary muscles. (B) Effect of temperature on maximal rate of force production (&SEM) in the three species. Values expressed in percentage of steady state values at 37°C.
0
542
LIU, WOHLFART,
AND JOHANSSON
A 25001
2000 I
Hedgehog
1
3
1200-
FE F 60&
1
II
35
30
25
I
I,
20
15
Temperature
IJ
10
5
I
0
35
30
1
I
I
I
25
20
15
10
Temperature
(4~)
J
5
0
(‘c)
FIG. 3. (A) Time from stimulus to peak contractile force (?SEM) at different temperatures in rabbit, rat, and hedgehog papillary muscles. (B) Time from peak force to half-relaxation (THR) related to temperature. Values expressed in percentage of steady state values at 37°C. Same experiments as in Fig. 2.
longed as can be seen in the Fig. 3B. The below 10°C as can be seen in Fig, 4 (at the percentage of prolongation was about the same time there was also an increased resting force). In contrast, there were no aftersame in the three different preparations. After-contractions were regularly re- contractions in the hedgehog preparations, corded in rabbit papillary muscles at a tem- even at temperatures close to 0°C. Another sign of calcium overload at low perature of 13°C (Fig. 4). At the same time there was an increase in resting force as temperatures might be inferred from Fig, 5. described above (not shown in the figure). Mechanical restitution curves were reIn the rat papillary muscles, after-contrac- corded at 15°C in the three species, i.e., tions occurred as temperature was reduced peak force of a test contraction was related Rabbit
lOmN] h Rat
iomNl AL
A----
Hedgehog
lomNl A--IL A...- J-L..-13 “C
5 “C
0 “C
FIG. 4. Isometric contractions of rabbit, rat, and hedgehog papillary muschs at different temperatures as indicated in the figure. After-contractions are seen in the rabbit papillary muscle at 13°C and in the rat papillary muscle at 5°C. No after-contractions were recorded in the hedgehog preparations.
HYPOTHERMIC
EFFECTS
ON PAPILLARY
MUSCLES
543
was also a similar prolongation in all three species of time to peak force and of time from peak to half-relaxation as the temperature was lowered. An increase of peak isometric force of mammalian myocardium was also observed in other studies when the temperature was decreased to about 20°C (5,14,33,41,48). In skinned Purkinje fibres 01 , , , I of canine, the sensitivity of the myofila0.5 1.0 2.5 10 30 120 600 ments to calcium strongly increased when Test interval (s) temperature was lowered from 22 to 12°C FIG. 5. Mechanical restitution curves of rabbit, rat, (17) and time to peak force was markedly and hedgehog papillary muscles at a temperature of prolonged. This prolongation might be 15°C. The preparations were basically paced at a frecaused by a slowing of temperature-sensiquency of 0.5 Hz. At regular times a test pulse was introduced after a varied interval and peak force of the tive chemical reactions subsequent to a test contraction (FI max) was analyzed. The three me- very rapid calcium binding to Troponin C chanical restitution curves were nomalized to their (25). It would also follow, to some extent, maximal values (F,,J, respectively. from the temperature effects on calcium release and reuptake from the sarcoplasmic to the duration of the preceding test pulse reticulum (9,46). In a concomitant study on interval. This interval was varied from 0.5 rabbit, rat, and hedgehog cardiac preparato 600 sec. As the interval was prolonged, tions, we reported that the mechanical resforce increased in all three preparations. titution started later at low temperatures Beyond about 90 set there was a decline in (37). force in the hedgehog ventricular muscle. The regulation of intracellular calcium is However, there was no decline in force at important in cardiac cells (28, 29). Afterlonger intervals in the rabbit and rat mus- contractions are considered to be a sign of cles. calcium overload in the cell (2, 26,49), Any interference with the ability to maintain a DISCUSSION low intracellular calcium concentration In the present study we have compared would result in after-contractions and inthe cardiac mechanical restitution and con- creased resting tension (22, 30-32, 45). So far, two mechanisms are known to be tractions in hibernating (hedgehog) and nonhibernating (rabbit, rat) species. Adult related to calcium extrusion and mainteanimals of all three species were used, nance of low calcium concentration in carsince it has been shown that cold tolerance diac cells. The first is the Na-Ca exchange in which an entry of Na is coupled to the changes with development of individuals (1, 19). The data obtained showed that the transport of Ca from cytoplasmic to extracardiac muscle of hedgehog still contracted cellular space (15, 34, 42, 44, 45). The enat O”C, while the cardiac muscles of rabbit ergy for this exchange is derived from the and rat developed after-contractions and in- Na-K pump which actively transports Na creased resting force below 15 and lO*C, out of the cell and so establishes the Narespectively, and were not excitable below gradient across the membrane (8, 45). The 10 and 5°C respectively. A reduction in second is the ATP-dependent calcium temperature was also associated with an in- pump in myocardial sarcolemma (6, 12,47). crease in peak developed force. The opti- With radioactive 4SCa measurements, the mum was seen at 20°C in the rabbit, 15°C in calcium efflux was inhibited at low temperthe rat, and 10°C in the hedgehog. There ature in guinea pig myocardium (44). In re-
544
LIU, WOHLFART,
cent studies with fresh red cells from hibernating species (hedgehogand ground squirrel) there was an active calcium efflux when the cells were incubated at 5°C. However, fresh red cells from guinea pig, or ATP depleted red cells from hedgehogand ground squirrel, showed a significant accumulation of intracellular calcium at low temperatures (16, 18). In the present study the following signs of cellular calcium accumulation in response to hypothermia were observed: (1) Increased resting force, (2) after-contractions, and (3) a reduced decline of force after the maximum in the mechanical restitution curve at the longest test intervals (Fig. 5). (This probably reflects a reduced cellular calcium outtlow.) Signs of calcium overload ((1) and (2) above) were seen at higher temperatures in the rabbit ventricular muscle as compared to that of the rat. No sign of calcium overload was seen in the hedgehog preparation. A rise of intracellular calcium above the normal diastolic value would activate a transient inward current (3, 10, 27, 35, 40). This might cause afterdepolarizations which could initiate extrasystoles and start severe arrhythmias in the heart (11). The greater cold tolerance of the hedgehog heart and its resistance to arrhythmias might to some extent be explained by the calcium handling system in the hedgehog myocardium preventing calcium overload at low temperatures. Further studies on calcium regulation in cardiac cells would provide more evidence for this hypothesis. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (14P-7920, 14X-184, 14X-08664)Ernhold Lundstriims Stiftelse and from the Faculty of Medicine, University of Lund. The stay in Sweden for Dr. Bin Liu was supported by the Swedish National Association against Heart and Chest Diseases. REFERENCES 1. Adolph, E. F. Responses to hypothermia in sev-
AND JOHANSSON eral species of infant mammals. Amer. J. Physiol. 166, 75-91 (1951). 2. Allen, D. G., Eisner, D. A., Pirolo, J. S., and Smith, G. L. The relationship between intracellular calcium and contraction in calciumoverloaded ferret papillary muscles. J. Physiol. 364, 169-182 (1985). 3. Arlock, P., and Katzung, B. G. Effects of sodium substitutes on transient inward current and tension in guinea-pig and ferret papillary muscle. J. Physiol. 360, 105-120 (1985). 4. Bi(irck, G., and Johansson, B. W. Comparative studies on temperature effects upon electrocardiogram in some vertebrates. Actu Physiol. Scand. 34, 257-272 (1955). 5. Blinks, J. R., and Koch-Weser, J. Physical factors in the analysis of the actions of drugs on myocardial contractility. Pharmacol. Rev. 15, 531600 (1%3). 6. Caroni, P., and Carafoli, E. An ATP-dependent Ca-pumping system on dog heart sarcolemma. Nature (London) 283, 765-767 (1980). 7. Chao, I., and Yeh, C. J. Temperature and activity of the excised perfused heart of the hedgehog. Chin. J. Physiol. 18, 17-30 (1951). 8. Chapman, R. A., Coray, A., and McCiuigan, J. A. S. Sodium/calcium exchange in mammalian ventricular muscle: study with sodiumsensitive microelectrodes. J. Physiol. 343, 253276 (1983). 9. Chiesi, M. Temperature-dependency of the functional activities of dog cardiac sarcoplasmic reticulum: a comparison with sarcoplasmic reticulum from rabbit and lobster muscle. J. Mol. Cell. Cardiol. 11, 245-259 (1979). IO. Colquhoun, D., Neher, E., Reuter, H., and Stevens, C. S. Inward current channels activated by intracellular calcium in cultured heart cells. Nature (London) 294, 752-754 (i981). I11. Cranefield, P. F. Action potentials, afterpotentials, and arrhythmias. Circ. Res. 41, 415-423 (1977). t2. Dhalla, N. S. Significance of Ca ATPases in the control of calcium movements in heart sarcolemma. J. Mol. Cell. Cardiol. 12, suppl I, No. 35 (1980). 13. Duker, G., Sjdquist, P-O., Svensson, O., Wohlfart, B., and Johansson, B. W. Hypothermic effects on cardiac action potentials: Difference between a hibernator, hedgehog and a nonhibemator, guinea-pig. In “Living in the Cold: Physiological and Biochemical Adaptations” (Helter, H. C., Mussachia, X. J., and Wang, L. C. H. Eds.), pp. 565-572 Elsevier, New York. 14. Edman, K. A. P., Mattiazzi, A. R., and Nilsson, E. The influence of temperature on the force-
HYPOTHERMIC
EFFECTS ON PAPILLARY
velocity relationship in rabbit papillary muscle. Stand. 90, 750-756 (1974). Eisner, D. A., and Lederer, W. J. Na-Ca exchange: stochiometry and electrogenicity. Amer. 1. Physiul. 248, C189-202 (1985). Ellory, J. C., and Hall, A. C. Ca” transport in hibernator and non-hibernator species red cell at low temperature. J. Physiol. 345, 148P (1983). Fabiato, A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J. Gem Physiol. 85, 247-289 (1985). Hall, A. C., Wolowyk, M. W., Wang, L. C. H., and Ellory, J. C. The effects of temperature on Ca transport in red cells from a hibernator (Spermophilus richardsonii). J. Therm. Biol. 12, 61-63 (1987). Hubbard, K. W., Harris, B. W., and Hilton, F. K. The effects of temperature on isolated hearts from neonatal through postweanling rats. Comp. Bbchem. Physiol. 70A, 491495 (1981). Hudson, J. W. Thermal sensitivity of isolatedperfused hearts from the ground squirrels, Citellus tereticandus and litellus tridecemlineatus. 2. Vergl. Physiologie 71, 342-349 (1971). Jacobs, H. K., and South, F. E. Effects of temperature on cardiac transmembrane potentials in hibernation. Amer. J. Physial. 203, 403409 (1976). Jensen, R. A., and Katzung, B. G. Simultaneously recorded oscillations in membrane potential and isometric contractile force from cardiac muscle. Nature (London) 217, 961-963 (1968). Johansson, B. W., BiBrck, G., Haeger, K., and Sjiistrtim B. Electrocardiographic observations on patients operated upon in hypothermia. Acta. h4ed. Stand. 155, 257-269 (1956). Johansson, B. W. The effect of aconitine, adrenaline and procain, and changes in the ionic concentration in the production of ventricular fibrillation in a hibernator (hedgehog) and a nonhibernator (guinea-pig) at different temperatures. Cardiologiu (Base0 43, 158-169 (1%3). Johnson, J. D., Charlton, S. C., and Potter, J. D. A fluorescence stopped flow analysis of Ca exchange with troponin C. J. Biool. Chem. 254, 3497-3507 (1979). Kass, R. S., Lederer, W. J., Tsien, R. W., and Weingart, R. Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibers. J. Physiol. 281, 187-208 (1978). Kass, R. S., and Tsien, R. W. Fluctuations in Actor Physiol.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34. 35.
36.
37.
38.
39.
40.
41.
MUSCLES
545
membrane current driven by intracellular calcium in cardiac Purkinje fibers. Biophys. J. 38, 259-269 (1982). Katz, A. M., and Tada, M. The “stone heart”: a challenge to the biochemist. Amer. J. Cardiol. 29, 578-580 (1972). Katz, A. M., and Reuter, H. Cellular calcium and cardiac cell death. Amer. J. Cardiol. 44, 188190 (1979). Kaufmann, R., Fleckenstein, A., and Antoni, H. Ursachen und Ausldsungsbedingungen von Myokard-Kontraktionen ohne Reguliires Aktionspotential. Pfliigers Arch. ges. Physiol. 278, 435-446 (1963). Kort, A. A., Lakatta, E. G., Marban, E., Stern, M. D., and Wier, W. G. Fluctuations in intracellular calcium concentrations and their effect on tonic tension in canine cardiac Purkinje fibers. J. Physiul. 367, 291-308 (1985). Lakatta, E. G., and Lappe, D. L. Diastolic scattered light fluctuations, resting force and twitch force in mammalian cardiac muscle. J. Physiol. 315,369-394 (1981). Langer, G. A., and Brady, A. J. The effect of temperature upon contraction and ionic exchange in rabbit ventricular myocardium. Relation to control of active state. J. Gerr. Physiol. 52, 682713 (1968). Lauger, G. A. Sodium-calcium exchange in the heart. Annu. Rev. Physiol. 44, 43M49 (1982). Lederer, W. J., and Tsien, R. W. Transient inward current underlying arrhythmogenic effects of cardiotonic steriods in Purkinje fibers. J. Physiol. 263, 75-100 (1976). Liu, B., Zhao, M., and Chao, I. Effect of cold on transmembrane potentials in cardiac cells of the hedgehog. J. Therm. Biul. l&77-80 (1987). Liu, B., Wohlfart, B., and Johansson, B. W. Mechanical restitution at different temperatures in papillary muscles from rabbit, rat, and hedgehog. CryobioIogy 27, (1990) Lyman, C. P., and Blinks, D. C. The effects of temperature on the isolated hearts of closely related hibernators and non-hibernators. J. Cell. Camp. Physiol. 54, 53-fj3 (1959). Marshall, J. M., and Willis, J. S. The effects of temperature on membrane potentials in isolated atria of the ground-squirrel, Citellus tridecemlineatus. J. Physiol. 164, 676 (1962). Matsuda, H., Noma, A., Kurachi, Y., and Irisawa, H. Transient depolarization and spontaneous voltage fluctuations in isolated single cells from guinea-pig ventricles. Circ. Res. 51, 142-151 (1982). Mattiazzi, A. R., and Nilsson, E. The influence of
546
LIU,
WOHLFART,
temperature on the time course of the mechanical activity in rabbit papillary muscle. Acm Physiol.
scnnd. 97, 310-318 (1974).
42. Mullins, L. J. A mechanism for Na/Ca transport. J. Gen. Physiol. 70, 681-695 (1977). 43. Nordrehaug, J. E. Sustained ventricular tibrillation in deep accidental hypothermia. Brii. Med. J. 284, 867-868 (1982). 44. Reuter, H., and Seitz, N. The dependence of calcium eftlux from cardiac muscle on temperature and external ion composition. J. Physiol. 195, 451-470 (1968). 45. Sheu, S. S., and Fozzard, H. Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development. J. Gen. Physiol. 80, 325-351 (1982).
AND JOHANSSON
46. Suko J. The effect of temperature on Ca’+uptake and Ca activated ATP hydrolysis by cardiac sarcoplasmic reticulum. Experientiu 29, 396 398 (1973). 47. Sulhake, P. V., and St Louis, P. T. Passive and active calcium fluxes across plasma membranes. Progr. Biophys. Med. Mol. Bioi. 35, 135-195 (1980). 48. Svensson, 0. L. O., Wohlfart, B., and Johansson, B. W. Hypothermic effects on action potential and force production of hedgehog and guinea pig papillary muscles. Cryobiology 25, 445450 (1988). 49. Tsien, R. W., Kass, R. S., and Weingart, R. Cellular and subcelhalr mechanisms of cardiac pacemaker oscillations. 1. Exp. Viol. 81, 205215 (1979).
