Excitation-contraction mechanisms of the quail heart are functional at temperatures near freezing point

Excitation-contraction mechanisms of the quail heart are functional at temperatures near freezing point

J. therm. Biol. Vol. 14, No. 2, pp. 63-66, 1989 Printed in Great Britain.All rights reserved 0306-4565/89 $3.00+0.00 Copyright © 1989PergamonPress pi...

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J. therm. Biol. Vol. 14, No. 2, pp. 63-66, 1989 Printed in Great Britain.All rights reserved

0306-4565/89 $3.00+0.00 Copyright © 1989PergamonPress pie

EXCITATION-CONTRACTION MECHANISMS OF THE QUAIL HEART ARE FUNCTIONAL AT TEMPERATURES NEAR FREEZING POINT R. TIRRI and A. TALO Laboratory of Animal Physiology, Department of Biology, University of Turku, 20500 Turku, Finland (Received 30 April 1988; accepted in revised form 11 June 1988)

Abstract--1. Both subeellular contractile waves and the electrically triggered twitches of the isolated cardiac myocytes of the quail (Coturnix coturnix japonica) could be observed down to 0°C, where contractions ceased but recovered if the temperature was raised before freezing took place. 2. The threshold for excitation to the electrical field stimulus increased steeply in single myocytes at temperatures below 20°C. Energy of activation (Ea) as calculated from Arrhenius plot for the rheobase values were 24 kJ above and 49 kJ below 20°C. 3. Electrical activity as well as visible contractions of the whole heart preparation stopped between 16 and 18°C,but could be triggered by electricalstimulus down to 6°C. However, irregular, spontaneous local contractions could still be observed in the sinus node area at 4°C. 4. Below,17°C the rheobase for the whole heart excitation increased more steeply than that of the single myocyte. 5. The results suggest that the excitation-contraction processes of the quail myocardial cellscan function down to the freezing point. Because of a failure in propagation of excitation in the syncytial structure the limit for the spontaneous beat of the whole heart lies between 16-18°C. Key Word Index--Excitation-contraction, heat temperature dependence, quail.


Single cardiac myocytes have been isolated enzymatically from different mammalian species (see e.g. Piper and Spieckermann, 1985a; 1985b). For the isolation of cells the tissue must be exposed to a low calcium concentration (10-50/~M added calcium). When the cells after isolation are reexposed to physiological calcium concentrations, a so called calcium paradox may develop. It means that the cells are unable to maintain their calcium homeostasis and become damaged (Zimmermann and Hulsmann, 1966; Hearse et al., 1978). If the isolation is successful, up to 90% of the single myocytes may be calcium-tolerant and stay undamaged. The enzymatical isolation technique has also been applied to adult bird (quail) heart to isolate single myocytes (Karttunen and Tirri, 1987) yielding about 50% of the cells with normal fine structure and tolerance to calcium. Like the mammalian cardiac myocytes most of the quail single myocytes are quiescent and could be induced to twitch by field stimulus. Some cells also show spontaneous twitches but more often they have subcellular contractile waves which propagate through the cells. The subcellular waves (phasic contractions) are more thoroughly studied on the mammalian myocytes. They usually initiate at the end of the cell and propagate slowly (100-200/zm/s) through the cell as a band of contraction which occupies 10-20% of the cell length. This process seems to be associated with the function of the sarcoplasmic reticulum (calcium-induced calcium release) rather than of sarcolemma since the waves also occur in cells in which the sarcolemma has been removed (Fabiato and Fabiato, 1972; Rieser et al., 1979). TB



The lower temperature limit for the heart beat of isolated adult non-hibernatinghomeothermic animals seems to be above 10°C while in hibernating species it is close to 0°C (Kuliabko, 1902; Lyman and Blinks, 1959; Covino and Hannon, 1959; Smith and Katzung, 1966; Nowell and White, 1967; Hubbard et al., 1981; Weller et al., 1978). Mechanically disaggregated mouse myocardial cells are able to contract spontaneously at 6°C (Bloom, 1970) and enzymatically isolated rat myocytes at 2°C (Vahony et al., 1970). We have found that both spontaneous subcellular waves and electrically triggered twitches can be observed in the rat myocytes even at 0°C (Tirri et al., 1983). The aim of the present study was (1) to determine, whether single cardiac myocytes of the non-hibernating bird could also tolerate such low temperatures, and (2) to compare the cold tolerance of single myocytes with the whole heart preparation. MATERIALS AND METHODS

Isolated myocytes

Quail of both sexes and 2-3 months of age were used. The method described by Karttunen and Tirri (1987) was used to isolate ventricular myocytes. A sample of cells suspended in 100-150 # 1physiological salt solution containing (in mmol/l) NaCI 137, KCI 4.6, MgCI 2 1.2, NaHPO 3.5, CaCI 2 1.0, glucose 10 and HEPES-NaOH 10 at pH 7.3, was placed on a cavity slide and observed under a microscope. The contractions of the cells were recorded on a videotape recorder (Sony SLC7E) and analysed in detail later. The test period lasted no more than 5 rain to avoid changes due to alteration of pH, dessication and oxygen conditions. Excitability of the myocytes was studied by



stimulating the cells by platinum field electrodes placed parallel to the cell under study. The values of the rheobase (i.e. the minimum effective voltage) were determined as previously described (Karttunen and Tirri, 1987).


°C 20






Whole heart preparation Heparinized quails were decapitated, the heart was removed and coronary perfusion with the physiological salt buffer (see above) was initiated in a Langendorf perfusion apparatus. After 10 min perfusion at 37°C the cold tolerance was measured by lowering the temperature at a rate of 0.3°C/min (Lauda K4R programmable thermostat). The cardiac action potential was recorded extracellularly by a Grass polygraph using flexible suction electrodes made of silicone tubing (inner diameter 1.5 mm) inside of which 50/~m diameter silver wire reached almost to the tip of the electrode. An electrode was placed on the surface of the atrium and another in to the middle of the ventricle. Negative pressure (1 3 mmHg) from a water outlet was connected into the electrodes through a bottle. This kept the electrodes sealed on the heart surface during contractions of the heart. After cessation of the spontaneous action potentials and contractions due to the lowered temperature, electrical stimulation was started. Stimulus pulses were delivered into the atria through a pair of the suction electrodes placed on the atrial surface, close to the sinus node. Additional experiments were carried out on isolated atria where the contractions of sinus node area and the inner surface of the atria were observed through a dissection microscope while simultaneously recording the electrical activity by small suction electrodes (inner diameter about 200 pm). The electrodes were placed on the sinus node and at various distances from it on muscle bundles originating from the nodal area. The preparation was incubated in the oxygenated physiological buffer solution and the temperature was varied.


E tO0


3 2



TIK 10'

Fig. I. Temperature dependence of the velocity of contraction waves expressed as an Arrhenius plot. The points are

means of 5-16 cells. Bars indicate ±SEM. Ea is the value of apparent activation energy as kJ mol. Between the isolation and experimentation the cells were maintained at 23c'C.

linearly temperature dependent and the value of the activation energy was 56 kJ. Because of the large variation of the frequency of the waves its temperature dependency was not analysed. Spontaneous twitch contractions occasionally seen at room temperature disappeared in the temperature range between 15 and 20°C. However, twitch contractions triggered by field stimulation and characterized by simultaneous sarcomere shortening throughout the cell could be observed down to the freezing point and they disappeared on average at - 0 . 3 + 0.27°C, n = 5. The excitation threshold for extracellular stimulus increased with the declining temperature. The rheobase values showed that the excitability started to drop more steeply when the temperature fell below 17°C (Fig. 2). Energy of activation as calculated from the Arrhenius plot for the rheobase values of 14 myocytes was 24 kJ at temperatures above 15°C and 49 kJ below 15°C.


Cold tolerance of the isolated single myocytes Enzymatical isolation of cardiac myocytes yielded rounded cells and morphologically intact, spindleshaped cells. When exposed to physiological levels of calcium some of the latter cells started to contract repeatedly and went into a sustained contracture. Most of them, however, were able to maintain their calcium homeostasis and morphology when exposed to physiological concentration of calcium. These myocytes were calcium-tolerant. In accordance with earlier observations by Karttunen and Tirri 0987) about 80% of the calcium-tolerant myocytes were quiescent at room temperature. The others had spontaneous subcellular waves (phasic contractions) which propagated through the cells. Spontaneous twitch contractions were also occasionally seen. Twitches could be triggered by the field electrodes in about 90% of the calcium-tolerant myocytes. Lowering the temperature did not abolish the subcellular waves until the temperature was about 0°C ( - 0 . 2 + 0.27°C, n = 7). The velocity of the subcellular contraction waves declined steeply with the falling temperature (Fig. 1). On the Arrhenius plot the decrease was

°C 30

± V




0.2 o





3.3 . .


. 3.4



3 .6

l .10 3 T°K

Fig. 2. Temperature dependence of the rheobase for single myocytes presented as reciprocals on an Arrhenius plot. Each point represents the mean of 8-14 cells. The inserted figure shows the original values on the linear scales. Vertical bars indicate + SEM. The distance between the stimulating electrodes was about 500 ~m.

Excitation-contraction mechanisms of quail heart CoM tolerance o f the whole isolated heart and isolated atria The temperature dependence of the perfused quail heart rate changed at about 22°C. On the Arrhenius plot the linear relationships above 22°C gave 37 kJ and below 22°C 79 kJ for values of the activation energies (Fig. 3). Electrical activity and visible contractile activity stopped between 16 and 18°C, (n = 7). The activity first ceased in the ventricles. When the heart was driven by electrical stimuli applied to the atria, contractions could be induced even at 6°C. Also in these cases contractions did not always spread through the ventricle at the lowest temperatures. The excitation threshold of the heart for the electrical stimulation increased drastically when lowering .the temperature (Fig. 4). The lower graph showing the reciprocals of the rheobase values on the Arrhenius plot gives a linear relationship with Ea 84 kJ. The temperature limit found in the present study for the electrically driven heart might be lower than 6°C because our stimulator gave a maximum of 80 V (100 ms) stimulus pulse while higher intensities might have induced contractions at even lower temperatures. In three atrial preparations the sinus node area and the muscle bundles on the atrial inner surface were observed under a dissection microscope, while recording the action potentials with small suction electrodes placed to the sinus and on the muscle bundles originating from it. Sporadic contractions were still observed and local electrical activity recorded in the nodal region at 4°C. This activity sometimes spread out for a short distance along a muscle bundle but did not extend through the whole atrium as it did in room temperature. DISCUSSION

SubeeUular contraction waves which occur in the cardiac myocytes are associated with spontaneous calcium release from the sarcoplasmic reticulum (Endo et al., 1970; Fabiato and Fabiato, 1978) and are not triggered by action potentials (Lehto et aL, 1983).




°C 25



E a • 37 KJ

100 .F.




T--~. 103

Fig. 3. Temperature dependence of the beat rate of the isolated perfused heart expressed as an Arrhenius plot. The points are values from 7 hearts. Bars indicate + SEM.

65 °C






o o

1 0.05

o Ea=84KJ I 10





3.5 1




Fig. 4. Temperature dependence of the rheobase for the triggered beat in the isolated, perfused whole heart below the temperature limit for the spontaneous heart beat, shown as reciprocals on an Arrhenius plot. The inserted figure shows the original values on the linear scales. The points represent mean values from 5 hearts. Vertical bars indicate + SEM.

The calcium release causes an inward current (Talo et al., 1986) which is attributed to a sodium-calcium exchange mechanism (Clusin et al., 1983; Lipp and Pott, 1988). This basic contractile mechanism is functionable at nearly freezing temperatures. The temperature dependence of the velocity of these subceilular waves was much the same in the rat (Tirri et al., 1983) and quail myocytes, i.e. both had a linear temperature relationship on the Arrhenius plot with activation energy values of 54 and 55 k J, respectively. Similarly, electrically-triggered twitches could be induced, even at 0°C in the myocytes of both these homeotherm species. However, the excitation threshold increased even more markedly in the quail than in the rat, when the temperature fell below 20°C. The spontaneous rate of the whole heart preparation of the immature rat has two break points in the Arrhenius plot according to Hubbard et al. (1981). In the bird, european starling, the break point is at 27°C with activation energies of 43 kJ above and 124 kJ below this temperature (Weller et al., 1978). Our whole heart preparation of the quail gave only one break point at about 20°C. The activation energy values were 37 and 79 kJ for the temperature ranges above and below 20°C, respectively. Our results suggest that myocardial cells of homeotherms unable to hibernate are capable of functioning at temperatures near freezing point, but the whole heart ceases beating at a much higher temperature. This difference between single cells and the whole heart could be due to temperature effects on the pacemaker area or on the propagation of excitation. The presence of spontaneous activity in the pacemaker area at temperatures well below 10°C, found in the present study, indicates that cessation of the ventricular beat at about 17°C is not due to a nonfunctioning pacemaker but to lack of propagation. This may be due to temperature effects on the sodium



a n d calcium currents which c o n t r i b u t e to the rapid upstroke of the cardiac action potential. The time to peak o f the sodium a n d calcium currents is highly t e m p e r a t u r e d e p e n d e n t as is also the calcium current amplitude (Cavalie et al., 1985; Colatsky, 1980). T h u s lowering of the t e m p e r a t u r e leads to a slowing a n d reduction of the i n w a r d currents. Since the inactive tissue forms an electrical load to the adjacent tissue undergoing excitation, this leads to a progressive reduction of the capability to form a p r o p a g a t i n g action potential in a syncytial tissue (Tan et al., 1988). This can be c o m p e n s a t e d by the increase of the total volume of the tissue undergoing excitation as we did by increasing the triggering stimulus intensity. This contrasts with the single myocyte which has no electrical load from the adjacent cells a n d therefore its excitation is less sensitive to the reduction o f the temperature. A c k n o w l e d g e m e n t s ~ u r thanks are due to Ms Sinikka Hillgren and Mr Veijo Kupari for their assistance.


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