CRYOBIOLOGY
28, 96-104 (1991)
Temperature
Effects on the Na and Ca Currents Hedgehog Ventricular Muscle
in Rat and
BIN LIU,’ PER ARLOCK, BJGRN WOHLFART, AND BENGT W. JOHANSSON Departments
of Zoophysiology and Pharmacology, University of Lund, S-223 62 Lund, and Section of Cardiology, General Hospital, S-214 01 Malmii, Sweden
Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35°C in both species resting potential was about - 80 mV and upstroke velocity (V,,,,,) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular ceils at 3X was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and V,,, declined. In rat ventricular cells at lO”C, the resting potential was about -40 to -50 mV and V,,, was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and e,,, were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20°C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10°C the availability curve of the Na current shifted toward more negative potentials and Zca,Fak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and Zca,reakdid not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20°C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators. B I-M Academic press, IIK
Na and Ca currents in hibernator (hedgehog) and nonhibernator (rat) ventricular muscle with the single sucrose-gap voltageclamp technique. At the same time, the change of transmembrane potentials with temperature was also compared between the two species.
Cold tolerance and resistance to ventricular fibrillation are two characteristics known in mammalian hibernators (22, 29). They rarely develop ventricular fibrillation not only in hibernation or hypothermia, but also in other situations in which ventricular fibrillation is easily induced in nonhibernators (12, 20). The electrophysiological studies in hibernators have shown that the conduction velocity of the heart is less influenced by cooling than in nonhibernators (14). Furthermore, the cardiac transmembrane potentials could be recorded at as low as 0°C (28). It is to be expected that the ion channels of the cardiac membranes are more resistant to lower temperatures in hibernators than in nonhibernators. In order to investigate this hypothesis, we have studied the
METHODS
Preparations. Ten rats (200 g, SpragueDawley) were sacrificed by cervical dislocation. The right ventricular papillary muscle (diameter 0.2-0.4 mm) was excised under a Zeiss stereo microscope and mounted in a single sucrose-gap apparatus as described previously (3). The tendinous tip of the papillary muscle in the test chamber was tied to a hook fastened to an AKER Electronics semiconductor strain-gauge by a short length of 6-O surgical silk suture. Received March 2,1989; accepted January 15,199O. Six hedgehogs (50&1100 g, Erinaceus europaeus) were trapped in the south of Swe’ Present address: Department of Zoology, University of Alberta, Edmonton, Canada. den in June and July. The animals were 96
001l-2240/91 $3.00 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
TEMPERATURE
EFFECTS
kept in a nonhibernating state at a photoperiod of LD 12:12 at 18°C and supplied with water and cat food (Mjau). The experiments on the hedgehogs were performed in September and October. The hedgehogs were anesthetized with thiopental sodium (30 mg/kg). The right papillary muscles were prepared as described in the rat. Solutions. The Tris-Tyrode solution contained (mM): NaCl 145, KC1 4.0, CaCl, 1.8, MgCl, 1.05, glucose 5.0 and Tris-HCl 5.0. The posterior (current-injecting) compartment was perfused with Tris-buffered solution as described above with NaCl replaced by KCl. The middle compartment was perfused with isotonic sucrose solution which contained (mM) sucrose 300, glucose 5.0, and CaCl, 0.04 (in some experiments with hedgehog papillary muscles, CaCl, 0.15 mM was used in sucrose-gap solution). All solutions were gassed with 100% 0,. The pH of the normal Tyrode and isotonic KC1 solutions was adjusted to 7.4. The temperature of the bath solution was monitored by means of a thermistor. Procedures. The papillary muscle preparation was held at 95% of optimum muscle length and equilibrated at a bath temperature of 35°C for 60 min. During this period all three compartments were perfused with normal Tyrode solution. The sucrose-gap was established by perfusing the middle compartment with isotonic sucrose solution. The muscle preparation was stimulated with a Grass S-4 stimulator at 0.3 Hz. The stimulus pulse was 3 ms duration at twice the threshold for excitation. During voltage clamp, the duration of the clamp step was 300-500 ms. The transmembrane potentials and currents were displayed on a digital storage oscilloscope (Tektronix 5223). In parallel the signals were recorded on an Elema-Schonander ink-jet chart recorder and via a Medical System Corporation sampling unit on a video tape with a Sony Video 8 videocassette recorder. In addition, photographs could be taken from another oscilloscope (Tektronix 502A). After the recordings at 35°C the bath
ON
Na AND
Ca CURRENTS
97
temperature was held at 20, 15, and 10°C for at least 30 min for equilibration. The same experimental procedure was used at each temperature. Measurements and statistics. Measurements of voltage and current were made on oscilloscope recordings or paper recordings of the signals. Student’s t test for paired data was used to analyze the differences of the correspondent values between rat and hedgehog. RESULTS
Temperature Effects on Transmembrane Potentials Figure 1 shows representative recordings of transmembrane potentials and V,,, at different temperatures in rat and hedgehog papillary muscle. Mean values and statistical analysis of these parameters are given in Table 1. In both preparations the amplitude of the action potential reached more than 100 mV and qm,, was above 100 V/s at 35°C. The shape of the action potential in hedgehog papillary muscle was similar to that of the rat lacking a distinct plateau phase at 35°C. When the temperature was decreased the membrane resting potential changed in a depolarizing direction, V,,,, declined, and the duration of action potential was increased. In hedgehog papillary muscle, the prolongation of phase 3 of the action potential was more marked below 20°C (P < 0.01) as can be seen in the figure. V,,,,, decreased to about 10 V/s at 10°C but the amplitude of the action potential was still maintained at a relatively high level, i.e., about 70-80 mV. In rat papillary muscle, the prolongation of action potential was limited in comparison with the hedgehog. Below 15°C the overshoot of the action potential disappeared and i/-maxwas only about 5 V/s. At this low temperature, the initial phase of the action potential was slow resembling a calcium action potential. Temperature Effects on Na Current The preparations were clamped to a hold-
98
LIU ET AL.
Iii-2
15oc
2o"c
35%
10°C
FIG. 1. Action potentials and the differentiated upstroke (Q,,) from a typical rat (A) and a hedgehog (B) papillary muscle at the different temperatures indicated in the figure.
ing potential between - 50 and - 140 mV in calcium-free solution. Following a depolarizing step the Na current was recorded at different temperatures between 20 and 10°C. In rat papillary muscle at 20°C the Na current could be recorded at a membrane holding potential up to - 60 mV (Fig. 2A). At a holding potential of -90 mV, the Na
channels could be completely activated by a depolarizing pulse. At lower temperatures the steady-state availability curve for the Na current was shifted toward more negative values as can be seen in the figure. The threshold membrane potential for the inactivation of the Na current was about &-7O mV at 15°C and -90 mV at 10°C. In unclamped modes, however, the rat cardiac
TABLE 1 Effects of Temperature on Resting Potential (RP), Action Potential Amplitude (APA) and Duration (APD), and Upstroke Velocity (V,,,) of Rat (R) and Hedgehog (HH) Ventricular Muscle APD (ms)
RP (mv) 35 20 15
10
50%
70%
90%
R HH
-83k2 -86k2
106 f 2 107 + 1
106 % 2 116 2 7
31 a 2** 17 * 1
42
31 ?I 2
61 -1- 5 54 + 1
9021
R HH
-8022 -8222
97 2 2 96 k 2
39 2 5* 60 * 2
51 r 2% 45 r 1
70" 2** 87 2 2
93 f 1** 169 * 2
273 2 3
R HH
-66 2 2* -755 1
80 k 2** 92 t 1
22 2 1** 42 2 1
74 -c 4**
51 It 1
106 2 6
221 2 2
179 +- 6** 366+-2
R HH
-48 + 3** -702 1
47 * 3** 79 2 1
5 2 1** 11 f 1
99k6 96 k 2
118 f 7** 157 k 4
161 2 9** 311 2 3
213 2 11** 514 * 3
2
3
99*4
122 2 6**
%-+4
161 2 lo**
Note. Means + SE. The difference in each paired data between the rat and hedgehog is marked in the table: *P < 0.05; **P < 0.01.
TEMPERATURE
EFFECTS
FIG. 2A. Steady-state availability curve of the Na current in rat papillary muscle at different temperatures 20°C (tilled circles), 15°C(open circles), and 10°C (triangles). The Na current was expressed as percentage of the maximum at each temperature.
membrane depolarized from - 75- - 85 mV at 35°C to -4O-- 50 mV at 10°C. This means that the Na channels in rat cardiac membrane were completely inactivated at 10°C when the resting potential was around -40 to -50 mV. Figure 2B shows the steady-state availability curve of the Na current in hedgehog papillary muscle. At 20°C the inactivation of Na channels was similar to that in the rat. However, the Na channels in hedgehog cardiac membrane were less influenced by the reduction of temperature. At lO”C, the Na current reached the maximum at about - 100 mV and was inactivated at potentials less than -75 mV. The cold tolerance of the sodium channel was thus greater in the hedgehog preparation in comparison with the rat. Temperature Effects on the Calcium Current
The inward Ca current could be recorded when the membrane holding potential was clamped at -40 mV, because at this voltage the Na channels are completely inactivated. In some control experiments Na current was inhibited by substituting Na with sucrose and Li in the Tyrode solution which gave the same results. Typical recordings of the Ca current in
ON
Na
AND
Ca
CURRENTS
99
FIG. 2B. Steady-state availability of the Na current in hedgehog papillary muscle. Same symbols as in Fig. 2A.
rat and hedgehog papillary muscle are shown in Figs. 3A and 3B, respectively. In rat papillary muscle, both the activation and inactivation of the Ca current were retarded and the peak value declined at lower temperatures. ZCa,reakat 10°C was only about 20% of that at 35°C. The activation and inactivation kinetics of the Ca current in hedgehog muscle (Fig. 3B) were also slowed by cooling. But ZCa,reakdid not change significantly as temperature was lowered to 10°C. A current-voltage curve for the calcium channel is presented in Figs. 4A and 4B. The peak of the current-voltage curve was displaced to more negative potentials in the hedgehog as compared to the rat. When temperature was decreased from 35 to 10°C the current-voltage relation curve shifted toward more negative values in both hedgehog and rat papillary muscles. The amplitude of the calcium current was reduced at low temperatures in the rat but remained constant in the hedgehog preparation. Transient Inward Current (TZ) in Rat Papillary Muscle
A transient inward current (TI) could regularly be recorded after the second inward Ca current in the rat but was never seen in hedgehog cardiac preparations at low tem-
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LIU ET AL.
3cPc
15%
10%
FIG. 3. Typical voltage clamp recordings using a 500 ms depolarizing step to different voltage levels
in order to obtain I,,,,, in rat (A) and hedgehog (B) papillary muscle at different temperatures. Upper panels display current and lower panels display voltage. The initial part of the current signal in each pane1represents the capacitive current and followed by inward current (downward deflection) and an outward current (upward deflection).
peratures. A TI in a rat papillary muscle is shown in Fig. 5. The TI was more pronounced at 15 and 10°C than at 20°C. DISCUSSION
Transmembrane Potentials in Rat and Hedgehog Ventricular Muscle
The cardiac ventricular muscle of the rat is different from the same tissue of other mammalian species. It shows a fast phase of repolarization in the action potential (8, 17). In comparison with rat and guinea pig ventricular cells, the peak magnitude of the Ca current is similar in the two species, but there is a relatively rapid decay of Ca current in rat ventricular cells (23). An early outward current exists in rat but not in guinea pig ventricular cells and is responsible for the fast repolarization phase in the action potential (24).
The hedgehog ventricular cell also lacks a plateau phase of the action potential. The same results have been reported in other studies (14, 28). The fast repolarization phase of the action potential in hedgehog and in rat ventricular cells could be caused by the same mechanism. At low temperatures, the resting potential and the amplitude of the action potential are better maintained in hedgehog than in rat ventricular cells (P < 0.01 at 10°C). The prolonged duration of the action potential is related to the temperature effects on I,, (see Fig. 4) and on the outward potassium current (19). A significant difference between the two species is that phase 3 of the action potential duration is markedly prolonged below 20°C in hedgehog but not in rat ventricular cells (P < 0.01). This might be due to the sustained existence of a Na-Ca exchange current (calcium efflux,
TEMPERATURE A
l 2.0
i
(IA
101
EFFECTS ON Na AND Ca CURRENTS
P
B
0 .
35%
0 WC
a
10%
.40
4B. Current-voltage relationship of the Ca current in hedgehog papillary muscle at 35°C (tilled circles), 15°C (open circles), and 10°C (triangles). FIG.
l-4.0
4A. Current-voltage relationship of I,-. in rat papillary muscle at 35°C (tilled circles), 15°C (open circles), and 10°C (triangles). FIG.
cooling than those of nonhibernators (rat, rabbit, and sheep). sodium influx), since this Na-Ca exchange This study showed that temperature incurrent has been observed in phase 3 of the fluences the activation and inactivation of action potential in rat ventricular cells (3 1). Zc, more in rat than in hedgehog cardiac ventricular cells. The peak values of the Ca Na and Ca Currents at current decreased in rat when temperature Low Temperatures was reduced to lO”C, while the peak values of the Ca current did not change signifiBecause of the difficulty of clamping fast cantly in hedgehog. At low temperature, and large currents in a multicellular prepathe shift of the Ca current-voltage relationration (5), no attempts were made to anaships to more negative potentials was oblyze the activation kinetics of the Na current or the instantaneous current-voltage relationships of the Na channels. The availability of the Na current at 20°C in rat and hedgehog papillary muscle was similar to 2uA that reported for rabbit Purkinje fibers, which shows a complete inactivation at -10 mV -60 mV and full activation at potentials more negative than -90 mV (7). The shift -40 mV of the availability curve of the Na current to n more negative potentials is also described 5OOms in sheep and rabbit Purkinje fibers (7, 11). FIG. 5. Transient inward current (TI) in rat papillary The Na channels in cardiac membranes of muscle following repolarization after a 500 ms depothe hedgehog are thus less influenced by larizing voltage clamp step (temperature lS°C).
1
IA
I--
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LIU ET AL.
served in both species. This might be due to increased cellular Ca [Ca*+]r and has been confirmed in later studies. The same shift has been reported by increasing external Ca*+ or reducing external Na + ; two interventions which are both expected to increase [Ca*+]r (6). The effects of low temperature on the calcium current are thus compatible with an increased intracellular calcium concentration. The increase in calcium concentration at low temperature seems to be greater in the rat ventricular cell as compared to the hedgehog since the amplitude of the calcium current was reduced in the rat but not in the hedgehog experiments (see below). Ventricular
Fibrillation
Reentry of excitation to circus movements and action potentials triggered repeatedly by afterdepolarizations are possible causes of ventricular fibrillation (9, 33, 36). The reentry phenomenon is mainly determined by a critical interplay of the size of the heart, refractory period, and conduction velocity. Slowed conduction is consid.ered to be the dominant factor in the cause of ventricular fibrillation in nonhibernators during hypothermia (13). Resistance of the hibernator heart to ventricular fibrillation cannot be explained by the size of the heart (21) or differences in the cardiac refractory period (12). The amplitude and the rate of depolarization of the action potential are two important factors determining the conduction velocity (34). This study showed that the Na channels in hedgehog cardiac membrane were less influenced by the reduction of temperature. These two parameters, AP and p,,,, are therefore better maintained in hedgehog ventricular cells between 20 and 15°C. This includes the critical body temperature range of the ventricular fibrillation in nonhibernators. Triggered activity is sustained by the afterdepolarizations (10). An abnormal increase of [Ca2+li could cause a transient inward current (TI) which is responsible for
the delayed afterdepolarizations (4, 26, 30). From this point of view, it seems important to maintain [Ca*+]r at a constant level in preventing triggered activity in the heart. The TI could be recorded in rat but not in hedgehog ventricular cells. This is compatible with a higher calcium concentration in the rat cardiac cell as compared to the hedgehog at low temperatures as discussed above. A previous study (27) also indicated that Ca overload took place at low temperatures in rabbit and rat cardiac muscle but not in a hedgehog cardiac preparation. An abnormal increase of [Ca*+]r has also been reported during cardiac ischemia and hypoxia (1, 35), and in patients with heart failure (16). Membrane Properties
and Cold Tolerance
The membrane properties are important in the maintenance of cardiac function at low temperature. When a hibernator enters hibernation, the degree of unsaturation of fatty acids increases and the fluid state of cardiac membrane is thought to be preserved (2, 32). The phase transition and change of the molecular organization of membrane lipids are different in ground squirrel and dog heart, which shows two transitions in cardiac membrane, one at 26°C and the other at 15°C in the dog, but only one at 26°C in the active ground squirrel (32). At low temperatures, the Na-K pump in the plasma membrane is inhibited in nonhibernators (15). This means that the ionic gradients cannot be maintained. However, the resistance to low temperatures of the Na-K pump has been observed in many tissues from hibernators (25, 37). All these features and adaptations could well contribute to a maintained resting membrane potential and the functions of the Na- and Cachannels in the cardiac membranes of the hibernators. The relationship between the biochemical adaptations to temperature of the cardiac membranes and other cardiac functions has to be further studied, as it has been shown in a previous study, that the
TEMPERATURE
EFFECTS
diet of unsaturated fat could improve the cold tolerance of the rat heart (18). ACKNOWLEDGMENTS
This work was supported by grants from Stiftelsen Lars Hiertas Minne, Swedish Natural Science Research Council No. B-BU 3748-102 & 108, National Swedish Board for Technical Development (STUF) No. KB 51281-4, Swedish National Association against Heart and Chest Diseases, and Swedish National Association for Heart and Lung Patients, and by grants from the Swedish Medical Research Council (Projects 14F07920, 14X-08664). REFERENCES
1. Allen, D. G., and Orchard, C. H. Myocardial contractile function during ischemia and hypoxia. Circ. Res. 60, 153-168 (1987). 2. Aloia, R., Pengelley, E., Bolen, J., and Rouser, G. Changes in phospholipid composition in hibernating ground squirrel, Citellus lateralis and their relationship to membrane function at reduced temperature. Lipids 9, 993-999 (1974). 3. Arlock, P. Electrophysiological effects of amperozide in papillary muscles from ferrets, guinea pigs, and rabbits. Pharmacol. Toxicol. 62, 184191 (1988). 4. 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. (London) 360, 105-120 (1985). 5. Beeler, G. W. Jr., and McGuigan, J. A. S. Voltage clamping of multicellular myocardial preparations, capabilities, and limitations of existing methods. Prog. Biophys. Mol. Biol. 34,219-254 (1978). 6. Beeler, G. W. Jr., and Reuter, H. Membrane calcium current in ventricular myocardial fibers. J. Physiol. (London) 207, 191-209 (1970). 7. Colatsky, T. J. Voltage clamp measurements of sodium channel properties in rabbit cardiac Purkinje fibers. J. Physiol (London) 305, 215234 (1980). 8. Coraboeuf, E., and Vassort, G. Effects of some inhibitors of ionic permeabilities on ventricular action potential and contraction of rat and guinea pig hearts. J. Electrocardiol. 1, 19-30 (1968). 9. Cranetield, P. F. Action potentials, afterpotentials, and arrhythmias. Circ. Res. 41, 415-423 (1977). 10. Cranefield, P. F., and Wit, A. L. Cardiac arrhythmias. Annu. Rev. Physiol. 41, 459-472 (1979). 11. Dudel, J., and Rudel, R. Voltage and time dependence of excitatory sodium current in cooled
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sheep Purkinje fibers. Pfluegers Arch. 315,136158 (1970). 12. Duker, G. D., Olsson, S-O., Hecht, N. H., Senturia, J. B., and Johansson, B. W. Ventricular fibrillation in hibernators and nonhibernators. Cryobiology 20, 407-420 (1983). 13. Duker, G. D., Sjoquist, P-O., and Johansson, B. W. Monophasic action potentials during induced hypothermia in the hedgehog and guineapig heart. Amer. J. Physiol. 253, H1083-1088 (1988). 14. Duker, G. D., Sjoquist, P-O., Svensson, O., Wohlfart, B., and Johansson, B. W. Hypothermic effects on cardiac action potentials, difference between a hibernator, hedgehog, and a nonhibernator, guinea pig. In “Living in the Cold, Physiological and Biochemical Adaptations” (H. C. Heller, X. J. Musacchia, and L. C. H. Wang, Eds.), pp. 565-572. Elsevier, New York, 1986. 15. Glitsch, H. G., and Pusch, H. On the temperature dependence of the Na pump in sheep Purkinje fibers. Pfuegers Arch. 402, 109-115 (1984). 16. Gwathmey, J. K., Copelas, L., Mackinnon, R., Schoen, F. J., Feldman, M. D., Grossman, W., and Morgan, J. P. Abnormal intracellular calcium handling in myocardium from patients with end-state heart failure. Circ. Res. 61, 7076 (1987). 17. Hoffman, B. F., and Cranefield, P. F. Electrophysiology of the Heart. McGraw-Hill, New York, 1960. 18. Huttunen, M., and Johansson, B. W. The influence of the dietary fat on the lethal temperature in the hypothermia rat. Acta. Physiol. Stand. 59, 7-11 (1963). 19. Isenberg, G., and Trautwein, W. Temperature sensitivity of outward current in cardiac Purkinje fibers. Pji’uegers Arch. 358, 225-234 (1975). 20. Johansson, B. W. The effect of aconitine, adrenaline, and procaine, and changes in the ionic concentration in the production of ventricular fibrillation in a hibernator (hedgehog) and a nonhibernator (guinea pig) at different temperatures. Cardiologia 43, 158-169 (1963). 21. Johansson, B. W. Cardiac responses in relation to heart size. Cryobiology 21, 627-636 (1984). 22. Johansson, B. W. Ventricular repolarization and fibrillation threshold in hibernating species. Eur. Heart J. 6(Suppl. D), 53-62 (1985). 23. Josephson, I. R., Sanchez-chapula, J., and Brown, A. M. A comparison of calcium currents in rat and guinea pig single ventricular cells. Circ. Res. 54, 144-156 (1984). 24. Josephson, I. R., Sanchez-chapula, J., and
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Brown, A. M. Early outward current in rat single ventricular cells. Circ. Res. 54, 157-162 (1984). Kamm, K. E., Zatzman, A. W., Jones, M. L., and South F. E. Maintenance of ion concentration gradients in the cold in aorta from rat and ground squirrel. Amer J. Physiol. 237, C17-22 (1979). Kass, R. S., and Tsien, R. W. Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers. Biophys. J. 38, 259-269 (1982). Liu, B., Wohlfart, B., and Johansson, B. W. Effects of low temperature on contraction in papillary muscles from rabbit, rat and hedgehog. Cryobiology 27, 539-546 (1990). Liu, B., Zhao, M., and Chao, I. Effect of cold on transmembrane potentials in cardiac cells of the hedgehog. J. Therm. Biol. 12, 77-80 (1987). Lyman, C. P., Willis, J. S., Malan, A., and Wang, L. C. H. Hibernation and Torpor in Mammals and Birds. Academic Press, New York, 1982. 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). Mitchell, M. R., Powell, T., Terrar, D. A., and
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34. 35.
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Twist, V. W. Possible association between an inward current and the late plateau of action potentials in ventricular cells isolated from rat heart. J. Physiol. (London) 351, 40p (1984). Raison, J. K., McMurchie, E. J., Charnock, J. S., and Gibson, R. A. Differences in the thermal behaviour of myocardial membranes relative to hibernation. Camp. Biochem. Physiol. 69B, 169-174 (1981). Rosen, M. R., and Reder, R. F. Does triggered activity have a role in the genesis of cardiac arrhythmias? Ann. Int. Med. 94, 794-801 (1981). Spear, J. F., and Moore, E. N. Mechanisms of cardiac arrhythmias. Annu. Rev. Physiol. 44, 485497 (1982). Steenbergen, C., Murphy, E., Levy, L., and London, R. E. Elevation of cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ. Res. 60, 700-707 (1987). Surawicz, B. Ventricular fibrillation. Amer. J. Curdiol. 28, 268-287 (1971). Willis, J. S., Ellory, J. C., and Wolowyk, M. W. Temperature sensitivity of the sodium pump in red cells from various hibernator and nonhibernator species. J. Comp. Physiol. 138, 4347 (1980).
28, 96-104 (1991)
Temperature
Effects on the Na and Ca Currents Hedgehog Ventricular Muscle
in Rat and
BIN LIU,’ PER ARLOCK, BJGRN WOHLFART, AND BENGT W. JOHANSSON Departments
of Zoophysiology and Pharmacology, University of Lund, S-223 62 Lund, and Section of Cardiology, General Hospital, S-214 01 Malmii, Sweden
Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35°C in both species resting potential was about - 80 mV and upstroke velocity (V,,,,,) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular ceils at 3X was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and V,,, declined. In rat ventricular cells at lO”C, the resting potential was about -40 to -50 mV and V,,, was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and e,,, were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20°C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10°C the availability curve of the Na current shifted toward more negative potentials and Zca,Fak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and Zca,reakdid not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20°C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators. B I-M Academic press, IIK
Na and Ca currents in hibernator (hedgehog) and nonhibernator (rat) ventricular muscle with the single sucrose-gap voltageclamp technique. At the same time, the change of transmembrane potentials with temperature was also compared between the two species.
Cold tolerance and resistance to ventricular fibrillation are two characteristics known in mammalian hibernators (22, 29). They rarely develop ventricular fibrillation not only in hibernation or hypothermia, but also in other situations in which ventricular fibrillation is easily induced in nonhibernators (12, 20). The electrophysiological studies in hibernators have shown that the conduction velocity of the heart is less influenced by cooling than in nonhibernators (14). Furthermore, the cardiac transmembrane potentials could be recorded at as low as 0°C (28). It is to be expected that the ion channels of the cardiac membranes are more resistant to lower temperatures in hibernators than in nonhibernators. In order to investigate this hypothesis, we have studied the
METHODS
Preparations. Ten rats (200 g, SpragueDawley) were sacrificed by cervical dislocation. The right ventricular papillary muscle (diameter 0.2-0.4 mm) was excised under a Zeiss stereo microscope and mounted in a single sucrose-gap apparatus as described previously (3). The tendinous tip of the papillary muscle in the test chamber was tied to a hook fastened to an AKER Electronics semiconductor strain-gauge by a short length of 6-O surgical silk suture. Received March 2,1989; accepted January 15,199O. Six hedgehogs (50&1100 g, Erinaceus europaeus) were trapped in the south of Swe’ Present address: Department of Zoology, University of Alberta, Edmonton, Canada. den in June and July. The animals were 96
001l-2240/91 $3.00 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
TEMPERATURE
EFFECTS
kept in a nonhibernating state at a photoperiod of LD 12:12 at 18°C and supplied with water and cat food (Mjau). The experiments on the hedgehogs were performed in September and October. The hedgehogs were anesthetized with thiopental sodium (30 mg/kg). The right papillary muscles were prepared as described in the rat. Solutions. The Tris-Tyrode solution contained (mM): NaCl 145, KC1 4.0, CaCl, 1.8, MgCl, 1.05, glucose 5.0 and Tris-HCl 5.0. The posterior (current-injecting) compartment was perfused with Tris-buffered solution as described above with NaCl replaced by KCl. The middle compartment was perfused with isotonic sucrose solution which contained (mM) sucrose 300, glucose 5.0, and CaCl, 0.04 (in some experiments with hedgehog papillary muscles, CaCl, 0.15 mM was used in sucrose-gap solution). All solutions were gassed with 100% 0,. The pH of the normal Tyrode and isotonic KC1 solutions was adjusted to 7.4. The temperature of the bath solution was monitored by means of a thermistor. Procedures. The papillary muscle preparation was held at 95% of optimum muscle length and equilibrated at a bath temperature of 35°C for 60 min. During this period all three compartments were perfused with normal Tyrode solution. The sucrose-gap was established by perfusing the middle compartment with isotonic sucrose solution. The muscle preparation was stimulated with a Grass S-4 stimulator at 0.3 Hz. The stimulus pulse was 3 ms duration at twice the threshold for excitation. During voltage clamp, the duration of the clamp step was 300-500 ms. The transmembrane potentials and currents were displayed on a digital storage oscilloscope (Tektronix 5223). In parallel the signals were recorded on an Elema-Schonander ink-jet chart recorder and via a Medical System Corporation sampling unit on a video tape with a Sony Video 8 videocassette recorder. In addition, photographs could be taken from another oscilloscope (Tektronix 502A). After the recordings at 35°C the bath
ON
Na AND
Ca CURRENTS
97
temperature was held at 20, 15, and 10°C for at least 30 min for equilibration. The same experimental procedure was used at each temperature. Measurements and statistics. Measurements of voltage and current were made on oscilloscope recordings or paper recordings of the signals. Student’s t test for paired data was used to analyze the differences of the correspondent values between rat and hedgehog. RESULTS
Temperature Effects on Transmembrane Potentials Figure 1 shows representative recordings of transmembrane potentials and V,,, at different temperatures in rat and hedgehog papillary muscle. Mean values and statistical analysis of these parameters are given in Table 1. In both preparations the amplitude of the action potential reached more than 100 mV and qm,, was above 100 V/s at 35°C. The shape of the action potential in hedgehog papillary muscle was similar to that of the rat lacking a distinct plateau phase at 35°C. When the temperature was decreased the membrane resting potential changed in a depolarizing direction, V,,,, declined, and the duration of action potential was increased. In hedgehog papillary muscle, the prolongation of phase 3 of the action potential was more marked below 20°C (P < 0.01) as can be seen in the figure. V,,,,, decreased to about 10 V/s at 10°C but the amplitude of the action potential was still maintained at a relatively high level, i.e., about 70-80 mV. In rat papillary muscle, the prolongation of action potential was limited in comparison with the hedgehog. Below 15°C the overshoot of the action potential disappeared and i/-maxwas only about 5 V/s. At this low temperature, the initial phase of the action potential was slow resembling a calcium action potential. Temperature Effects on Na Current The preparations were clamped to a hold-
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Iii-2
15oc
2o"c
35%
10°C
FIG. 1. Action potentials and the differentiated upstroke (Q,,) from a typical rat (A) and a hedgehog (B) papillary muscle at the different temperatures indicated in the figure.
ing potential between - 50 and - 140 mV in calcium-free solution. Following a depolarizing step the Na current was recorded at different temperatures between 20 and 10°C. In rat papillary muscle at 20°C the Na current could be recorded at a membrane holding potential up to - 60 mV (Fig. 2A). At a holding potential of -90 mV, the Na
channels could be completely activated by a depolarizing pulse. At lower temperatures the steady-state availability curve for the Na current was shifted toward more negative values as can be seen in the figure. The threshold membrane potential for the inactivation of the Na current was about &-7O mV at 15°C and -90 mV at 10°C. In unclamped modes, however, the rat cardiac
TABLE 1 Effects of Temperature on Resting Potential (RP), Action Potential Amplitude (APA) and Duration (APD), and Upstroke Velocity (V,,,) of Rat (R) and Hedgehog (HH) Ventricular Muscle APD (ms)
RP (mv) 35 20 15
10
50%
70%
90%
R HH
-83k2 -86k2
106 f 2 107 + 1
106 % 2 116 2 7
31 a 2** 17 * 1
42
31 ?I 2
61 -1- 5 54 + 1
9021
R HH
-8022 -8222
97 2 2 96 k 2
39 2 5* 60 * 2
51 r 2% 45 r 1
70" 2** 87 2 2
93 f 1** 169 * 2
273 2 3
R HH
-66 2 2* -755 1
80 k 2** 92 t 1
22 2 1** 42 2 1
74 -c 4**
51 It 1
106 2 6
221 2 2
179 +- 6** 366+-2
R HH
-48 + 3** -702 1
47 * 3** 79 2 1
5 2 1** 11 f 1
99k6 96 k 2
118 f 7** 157 k 4
161 2 9** 311 2 3
213 2 11** 514 * 3
2
3
99*4
122 2 6**
%-+4
161 2 lo**
Note. Means + SE. The difference in each paired data between the rat and hedgehog is marked in the table: *P < 0.05; **P < 0.01.
TEMPERATURE
EFFECTS
FIG. 2A. Steady-state availability curve of the Na current in rat papillary muscle at different temperatures 20°C (tilled circles), 15°C(open circles), and 10°C (triangles). The Na current was expressed as percentage of the maximum at each temperature.
membrane depolarized from - 75- - 85 mV at 35°C to -4O-- 50 mV at 10°C. This means that the Na channels in rat cardiac membrane were completely inactivated at 10°C when the resting potential was around -40 to -50 mV. Figure 2B shows the steady-state availability curve of the Na current in hedgehog papillary muscle. At 20°C the inactivation of Na channels was similar to that in the rat. However, the Na channels in hedgehog cardiac membrane were less influenced by the reduction of temperature. At lO”C, the Na current reached the maximum at about - 100 mV and was inactivated at potentials less than -75 mV. The cold tolerance of the sodium channel was thus greater in the hedgehog preparation in comparison with the rat. Temperature Effects on the Calcium Current
The inward Ca current could be recorded when the membrane holding potential was clamped at -40 mV, because at this voltage the Na channels are completely inactivated. In some control experiments Na current was inhibited by substituting Na with sucrose and Li in the Tyrode solution which gave the same results. Typical recordings of the Ca current in
ON
Na
AND
Ca
CURRENTS
99
FIG. 2B. Steady-state availability of the Na current in hedgehog papillary muscle. Same symbols as in Fig. 2A.
rat and hedgehog papillary muscle are shown in Figs. 3A and 3B, respectively. In rat papillary muscle, both the activation and inactivation of the Ca current were retarded and the peak value declined at lower temperatures. ZCa,reakat 10°C was only about 20% of that at 35°C. The activation and inactivation kinetics of the Ca current in hedgehog muscle (Fig. 3B) were also slowed by cooling. But ZCa,reakdid not change significantly as temperature was lowered to 10°C. A current-voltage curve for the calcium channel is presented in Figs. 4A and 4B. The peak of the current-voltage curve was displaced to more negative potentials in the hedgehog as compared to the rat. When temperature was decreased from 35 to 10°C the current-voltage relation curve shifted toward more negative values in both hedgehog and rat papillary muscles. The amplitude of the calcium current was reduced at low temperatures in the rat but remained constant in the hedgehog preparation. Transient Inward Current (TZ) in Rat Papillary Muscle
A transient inward current (TI) could regularly be recorded after the second inward Ca current in the rat but was never seen in hedgehog cardiac preparations at low tem-
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3cPc
15%
10%
FIG. 3. Typical voltage clamp recordings using a 500 ms depolarizing step to different voltage levels
in order to obtain I,,,,, in rat (A) and hedgehog (B) papillary muscle at different temperatures. Upper panels display current and lower panels display voltage. The initial part of the current signal in each pane1represents the capacitive current and followed by inward current (downward deflection) and an outward current (upward deflection).
peratures. A TI in a rat papillary muscle is shown in Fig. 5. The TI was more pronounced at 15 and 10°C than at 20°C. DISCUSSION
Transmembrane Potentials in Rat and Hedgehog Ventricular Muscle
The cardiac ventricular muscle of the rat is different from the same tissue of other mammalian species. It shows a fast phase of repolarization in the action potential (8, 17). In comparison with rat and guinea pig ventricular cells, the peak magnitude of the Ca current is similar in the two species, but there is a relatively rapid decay of Ca current in rat ventricular cells (23). An early outward current exists in rat but not in guinea pig ventricular cells and is responsible for the fast repolarization phase in the action potential (24).
The hedgehog ventricular cell also lacks a plateau phase of the action potential. The same results have been reported in other studies (14, 28). The fast repolarization phase of the action potential in hedgehog and in rat ventricular cells could be caused by the same mechanism. At low temperatures, the resting potential and the amplitude of the action potential are better maintained in hedgehog than in rat ventricular cells (P < 0.01 at 10°C). The prolonged duration of the action potential is related to the temperature effects on I,, (see Fig. 4) and on the outward potassium current (19). A significant difference between the two species is that phase 3 of the action potential duration is markedly prolonged below 20°C in hedgehog but not in rat ventricular cells (P < 0.01). This might be due to the sustained existence of a Na-Ca exchange current (calcium efflux,
TEMPERATURE A
l 2.0
i
(IA
101
EFFECTS ON Na AND Ca CURRENTS
P
B
0 .
35%
0 WC
a
10%
.40
4B. Current-voltage relationship of the Ca current in hedgehog papillary muscle at 35°C (tilled circles), 15°C (open circles), and 10°C (triangles). FIG.
l-4.0
4A. Current-voltage relationship of I,-. in rat papillary muscle at 35°C (tilled circles), 15°C (open circles), and 10°C (triangles). FIG.
cooling than those of nonhibernators (rat, rabbit, and sheep). sodium influx), since this Na-Ca exchange This study showed that temperature incurrent has been observed in phase 3 of the fluences the activation and inactivation of action potential in rat ventricular cells (3 1). Zc, more in rat than in hedgehog cardiac ventricular cells. The peak values of the Ca Na and Ca Currents at current decreased in rat when temperature Low Temperatures was reduced to lO”C, while the peak values of the Ca current did not change signifiBecause of the difficulty of clamping fast cantly in hedgehog. At low temperature, and large currents in a multicellular prepathe shift of the Ca current-voltage relationration (5), no attempts were made to anaships to more negative potentials was oblyze the activation kinetics of the Na current or the instantaneous current-voltage relationships of the Na channels. The availability of the Na current at 20°C in rat and hedgehog papillary muscle was similar to 2uA that reported for rabbit Purkinje fibers, which shows a complete inactivation at -10 mV -60 mV and full activation at potentials more negative than -90 mV (7). The shift -40 mV of the availability curve of the Na current to n more negative potentials is also described 5OOms in sheep and rabbit Purkinje fibers (7, 11). FIG. 5. Transient inward current (TI) in rat papillary The Na channels in cardiac membranes of muscle following repolarization after a 500 ms depothe hedgehog are thus less influenced by larizing voltage clamp step (temperature lS°C).
1
IA
I--
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LIU ET AL.
served in both species. This might be due to increased cellular Ca [Ca*+]r and has been confirmed in later studies. The same shift has been reported by increasing external Ca*+ or reducing external Na + ; two interventions which are both expected to increase [Ca*+]r (6). The effects of low temperature on the calcium current are thus compatible with an increased intracellular calcium concentration. The increase in calcium concentration at low temperature seems to be greater in the rat ventricular cell as compared to the hedgehog since the amplitude of the calcium current was reduced in the rat but not in the hedgehog experiments (see below). Ventricular
Fibrillation
Reentry of excitation to circus movements and action potentials triggered repeatedly by afterdepolarizations are possible causes of ventricular fibrillation (9, 33, 36). The reentry phenomenon is mainly determined by a critical interplay of the size of the heart, refractory period, and conduction velocity. Slowed conduction is consid.ered to be the dominant factor in the cause of ventricular fibrillation in nonhibernators during hypothermia (13). Resistance of the hibernator heart to ventricular fibrillation cannot be explained by the size of the heart (21) or differences in the cardiac refractory period (12). The amplitude and the rate of depolarization of the action potential are two important factors determining the conduction velocity (34). This study showed that the Na channels in hedgehog cardiac membrane were less influenced by the reduction of temperature. These two parameters, AP and p,,,, are therefore better maintained in hedgehog ventricular cells between 20 and 15°C. This includes the critical body temperature range of the ventricular fibrillation in nonhibernators. Triggered activity is sustained by the afterdepolarizations (10). An abnormal increase of [Ca2+li could cause a transient inward current (TI) which is responsible for
the delayed afterdepolarizations (4, 26, 30). From this point of view, it seems important to maintain [Ca*+]r at a constant level in preventing triggered activity in the heart. The TI could be recorded in rat but not in hedgehog ventricular cells. This is compatible with a higher calcium concentration in the rat cardiac cell as compared to the hedgehog at low temperatures as discussed above. A previous study (27) also indicated that Ca overload took place at low temperatures in rabbit and rat cardiac muscle but not in a hedgehog cardiac preparation. An abnormal increase of [Ca*+]r has also been reported during cardiac ischemia and hypoxia (1, 35), and in patients with heart failure (16). Membrane Properties
and Cold Tolerance
The membrane properties are important in the maintenance of cardiac function at low temperature. When a hibernator enters hibernation, the degree of unsaturation of fatty acids increases and the fluid state of cardiac membrane is thought to be preserved (2, 32). The phase transition and change of the molecular organization of membrane lipids are different in ground squirrel and dog heart, which shows two transitions in cardiac membrane, one at 26°C and the other at 15°C in the dog, but only one at 26°C in the active ground squirrel (32). At low temperatures, the Na-K pump in the plasma membrane is inhibited in nonhibernators (15). This means that the ionic gradients cannot be maintained. However, the resistance to low temperatures of the Na-K pump has been observed in many tissues from hibernators (25, 37). All these features and adaptations could well contribute to a maintained resting membrane potential and the functions of the Na- and Cachannels in the cardiac membranes of the hibernators. The relationship between the biochemical adaptations to temperature of the cardiac membranes and other cardiac functions has to be further studied, as it has been shown in a previous study, that the
TEMPERATURE
EFFECTS
diet of unsaturated fat could improve the cold tolerance of the rat heart (18). ACKNOWLEDGMENTS
This work was supported by grants from Stiftelsen Lars Hiertas Minne, Swedish Natural Science Research Council No. B-BU 3748-102 & 108, National Swedish Board for Technical Development (STUF) No. KB 51281-4, Swedish National Association against Heart and Chest Diseases, and Swedish National Association for Heart and Lung Patients, and by grants from the Swedish Medical Research Council (Projects 14F07920, 14X-08664). REFERENCES
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