INTRODUCTION

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THE TELEOST HEART is working at temperatures that are cardioplegic in the mammalian heart. Furthermore, low temperatures have been reported to open the sarcoplasmic reticulum (SR) Ca²⁺ release channels (2), and a rapid lowering of temperature releases the SR Ca²⁺ content in the mammalian heart (2, 3). This, together with the difference in the morphology of cardiac myocytes (18), has been taken as evidence in favor of a more predominant role of the transsarcolemmal Ca²⁺ flux in the regulation of contraction in the lower vertebrate heart (see Refs. 5 and 21 for reviews). In accordance with this, studies using pharmacological manipulations of the SR function in multicellular preparations (7, 16, 20), together with measurements of ionic currents (23-25), suggest that the importance of the SR in the regulation of contraction is minor in teleost hearts at physiological temperatures. In contrast to this, measurements of the Na⁺/Ca²⁺ exchange (NCX) rate (13) and SR Ca²⁺ uptake and release (9, 10) indicate that the SR, despite a potent NCX, may contribute up to 50% of the total Ca²⁺ transient in the trout and toad heart (10, 11, 14), whereas Ca²⁺ influx through L-type Ca²⁺ channels at most can contribute 25% of the Ca²⁺ required to activate contraction at room temperature (11, 12). Furthermore, Ca²⁺ entry through L-type Ca²⁺ channels has been reported not to change significantly when the experimental temperature is changed (17) or by acclimatizing fish at different temperatures (24), whereas Ca²⁺ leak from the SR, contrary to the mammalian heart, has been reported to decrease when temperature is lowered in suspensions of trout ventricular myocytes (9). Additionally, acclimation at low temperatures has been reported to increase the SR Ca²⁺ uptake rate in permeabilized trout myocyte suspensions (1). The role of the SR in the regulation of contraction in the intact myocyte at low temperatures is, however, still an open question. To address this, we have used the perforated patch-clamp technique combined with rapid caffeine (Caf) applications and specific stimulation protocols that allow a determination of the accumulation (9, 11) and liberation of Ca²⁺ in the SR (10) at 21 and at 7°C.

	METHODS

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Trout atrial myocytes were isolated as previously described (12). Ionic currents were measured with the perforated patch configuration of the patch-clamp technique using a software-driven patch-clamp amplifier (EPC-9, HEKA Elektronic, Lambrecht, Germany). Cells were placed in front of one of five capillaries with the desired experimental solution. The flow rate was ~1 cm/s during experiments to avoid temperature gradients at the tip of the perfusion capillaries and to ensure rapid change of the extracellular solution. The experimental temperature was controlled (to ±1°C of the temperature setting) by countercurrent superfusion of the five capillaries containing the experimental solutions. The composition of the internal medium was (in mM): 100 CsCl, 3.1 Na₂ATP, 4 MgCl₂, 5 Na₂ phosphocreatine, 0.42 Li₂ guanosine 5'-triphosphate, 0.025 EGTA, 10 HEPES, and 20 tetraethylammonium (TEA). pH was adjusted to 7.2 with CsOH. Two-hundred-fifty micrograms per milliliters of freshly prepared amphotericin B were included in the pipette solution, and experiments were begun when the access resistance had decreased to less than four to five times the pipette resistance and remained stable (pipette resistance was 1-5 M). A sudden drop in the access resistance was assumed to reflect a rupture of the membrane patch, and cells showing this phenomenon were discarded. The external medium contained (in mM): 107 NaCl, 20 CsCl, 1.8 MgCl₂, 4 NaHCO₃, 0.8 NaH₂PO₄, 1.8 CaCl₂, 10 HEPES, 5 glucose, and 5 pyruvate. In some experiments, the cell was exposed to an external solution containing 10 mM TEA and/or 10 µM SITS to verify that no contaminating potassium or chloride currents were present. pH was adjusted to 7.4 at room temperature.

To determine SR Ca²⁺ uptake, its Ca²⁺ content was first cleared with a brief exposure to 10 mM Caf, and uptake was then induced by depolarizing the cell to different membrane potentials (V_m) for 10 s (see Ref. 9 for more details). The time integral of the tail current elicited with repolarization to 80 mV (labeled tail in Fig. 1A) was used as an index of the increase in total cytosolic [Ca²⁺] at the end of the depolarization above the total diastolic [Ca²⁺]. The amount of Ca²⁺ accumulated in the SR was determined from the NCX current elicited by exposing the cell to 10 mM Caf after repolarizing the cell to 80 mV (labeled Caf in Fig. 1A).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of temperature on sarcoplasmic reticulum (SR) Ca2+ loading. A: stimulation protocol. After clearing the SR Ca2+ content, the cell was depolarized to different membrane potentials (Vm) for 10 s to induce SR Ca2+ loading. After decay of the tail current (tail) elicited by returning Vm to 80 mV, caffeine (Caf) was applied to release the Ca2+ accumulated in the SR during the depolarization. B: tail currents elicited by returning Vm to 80 mV after a 10-s depolarization to the Vm given below each panel. The time scale represents 0.5 s. C: currents induced by rapid Caf exposure after a 10-s depolarization to the Vm given above each panel. The time scale represents 2 s.  Represent currents recorded at 21°C, and  represent currents recorded at 7°C.

To determine the amount of Ca²⁺ released from the SR, 10-ms depolarizations to different V_m were used, and the amount of Ca²⁺ liberated from the SR was determined from the time integral of the Caf-sensitive tail current elicited by repolarizing the cell to 80 mV. The Caf-sensitive tail current was obtained by subtracting the tail current immediately after exposing the cell to Caf from the corresponding tail current obtained with a loaded SR (see Ref. 10 for details). The SR load in these experiments was obtained from the time integral of the Caf-induced NCX current (see Ref. 10). With the use of the perforated patch configuration, only small or no changes were observed in the SR Ca²⁺ uptake and/or release with time (see also Ref. 9). Experiments were, nevertheless, begun at either 21 or 7°C to avoid possible time-dependent changes in the parameters examined.

Results are given as means ± SE, and statistical significance was evaluated with Student's t-test or ANOVA and Bonferroni posttest.

	RESULTS

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Figure 1A shows the stimulation protocol used to determine Ca²⁺ accumulation in the SR. Figure 1B shows tail currents elicited with repolarization from 30, 10, and +10 mV, and Fig. 1C shows the corresponding Caf-induced NCX currents. Current traces obtained at 21 and 7°C are superimposed for each V_m. The corresponding Caf-induced NCX currents are shown in Fig. 1C. As expected (see Ref. 11), both the tail current and the Caf-induced NCX current increased after depolarizations to more positive V_m. Figure 2A summarizes the dependency of the SR Ca²⁺ accumulation on the V_m at 21 and 7°C. In overall, temperature did not significantly affect the relationship between V_m and SR Ca²⁺ accumulation. This was also true when the SR was loaded by stimulating the cell with 20 consecutive 300-ms depolarizations at a frequency of 0.5 Hz (see Fig. 4B). Figure 2B shows that also the relationship between the time integral of the tail current and the time integral of the Caf-induced NCX current was not significantly different at 21 and 7°C. Pooling data at 21 and 7°C and fitting with a hyperbolic equation gave a maximal uptake of 66 amol/pF and a K_0.5 of 4.3 amol/pF.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Dependency of SR Ca2+ loading on Vm and cytosolic Ca2+ at 21 and 7°C. A: voltage dependency of SR Ca2+ accumulation during a 10-s depolarization at 21 () or 7°C (). Values are average of 8 to 9 cells. B: dependency of SR Ca2+ accumulation on the time integral of the tail current (Itail dt) at 21 () and 7°C (). The solid line was obtained by fitting the data with a hyperbolic equation.

Figure 3A shows representative Caf-sensitive current traces elicited by depolarizing the cell to 10, +10, +30, and +50 mV for 10 ms followed by repolarization to 80 mV. Current traces obtained at 21 and 7°C are superimposed. Figure 3B summarizes the relationship between V_m and the time integral of the Caf-sensitive tail current at 21 and 7°C. Values were obtained from 10 cells that had similar SR Ca²⁺ load at the two experimental temperatures (30.1 ± 5.5 and 31.2 ± 5.7 amol/pF at 21 and 7°C). No significant effect of temperature was found. Figure 3C shows the corresponding relationship between the SR Ca²⁺ content liberated with Caf and the amount of Ca²⁺ carried by the Caf-sensitive tail current at +10 mV. These results show that the SR both accumulates and liberates amounts of Ca²⁺ at 7°C that are similar to values found at room temperature.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of temperature on SR Ca2+ release. A: representative Caf-sensitive tail currents obtained by repolarizing the cell to 80 mV after a 10-ms depolarization to the Vm given above each panel. Time scale represents 250 ms. B: SR Ca2+ release was measured as the time integral of the Caf-sensitive tail current and plotted against the Vm during depolarization (n = 10). C: relationship between SR Ca2+ release and the Caf-releasable SR Ca2+ content (n = 10).  Were obtained at 21°C and  at 7°C.

To determine the relative importance of the SR in conditions that are closer to steady-state conditions, stimulation protocols using repetitive depolarizations were also used. Figure 4A shows superimposed current traces obtained on the first and the 20th depolarization of the cell to +10 mV after clearing the SR Ca²⁺ content with 10 mM Caf at the two experimental temperatures. Notice that clearing the SR Ca²⁺ content with 10 mM Caf slowed down inactivation of L-type Ca²⁺ current at both 21 and 7°C. At 21°C, the fast time constant decreased significantly from 15.0 ± 2.0 ms on the first depolarization to 10.4 ± 1.9 ms on the 20th depolarization (P < 0.01, n = 7). The corresponding values for 7°C were 73 ± 24 and 38 ± 15 (P < 0.01, n = 7) for the first and the 20th depolarization after clearance of the SR Ca²⁺ content. To evaluate the relative importance of sarcolemmal Ca²⁺ transport and Ca²⁺ accumulation in the SR at the two experimental temperatures, the SR Ca²⁺ content and the cumulative sarcolemmal Ca²⁺ extrusion after 20 depolarizations were also measured in 13 cells. Figure 4B shows the SR Ca²⁺ accumulation after 20 depolarizations to 30, 10, or +10 mV at a stimulation frequency of 0.5 Hz, and Fig. 4C shows the corresponding cumulative sarcolemmal Ca²⁺ extrusion. Temperature did not significantly affect the relationship between V_m and either SR Ca²⁺ content or cumulative sarcolemmal Ca²⁺ extrusion.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of temperature on SR function during repetitive stimulation. A: representative current traces elicited by depolarizing the cell from 80 to +10 mV for 300 ms at 21 (left) and 7°C (right). Superimposed traces were recorded on the first () and the 20th depolarization () after clearing the SR Ca2+ content. Only the first 150 ms of the depolarization are shown. B: relationship between Vm and SR Ca2+ load after 20 depolarizations. C: relationship between Vm and cumulative sarcolemmal Ca2+ extrusion after 20 depolarizations. Filled bars in B and C were obtained from 13 cells at 21°C, and open bars were obtained at 7°C.

	DISCUSSION

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The main finding of the present study is that the SR both accumulates and liberates Ca²⁺ at 7°C in trout atrial myocytes. Indeed, the stimulation protocols used to determine accumulation and liberation of Ca²⁺ from the SR show that temperature does not affect these parameters or their dependency on V_m. This is contrary to results from the mammalian heart where low temperatures have been shown to open the SR Ca²⁺ release channels and thus deplete the SR Ca²⁺ content (2). Furthermore, the results show that the relative importance of the SR in the regulation of contraction does not appear to diminish when temperature is lowered.

Dependency of SR Ca²+ uptake on temperature. The results in Figs. 1 and 2 show that the SR accumulates calcium equally well at 21 and at 7°C. The amount of Ca²⁺ accumulated during a 10-s depolarization to +10 mV gave average uptake rates of 3.3 and 3.5 amol · pF¹ · s¹ at 21 and 7°C, respectively. This is similar to the SR Ca²⁺ uptake rate previously reported for trout ventricular myocytes at a loading potential of +10 mV (9), and with a maximal uptake rate of 6.6 amol · pF¹ · s¹, the SR has the capacity to resequester a total Ca²⁺ transient of 4 amol/pF in 600 ms, corresponding to a rate of 100 beats/s at both temperatures. Considering that there is a strong loading of the SR during a 10-s depolarization to +10 mV, the average uptake rate is likely to be underestimated (see Ref. 9) and should be considered a lower estimate. In other words, the SR has the capacity to resequester the entire Ca²⁺ transient at physiological heart rates at both 21 and 7°C. Because the action potential duration is increased (16) and the NCX activity has been reported to decrease (15, 22) when temperature is lowered, the relative importance of SR Ca²⁺ uptake could be larger at 7 than at 21°C. Comparison of SR Ca²⁺ accumulation and cumulative sarcolemmal Ca²⁺ extrusion did not, however, show any significant effect of temperature on the relative importance of the two parameters. Temperature also did not significantly affect the relationship between the increase in total cytosolic [Ca²⁺] and SR Ca²⁺ uptake (assuming that the time integral of the tail current elicited with repolarization from a 10-s depolarization reflects the increase in the total cytosolic [Ca²⁺] at the end of the depolarization above the total resting [Ca²⁺]). The exact relationship between SR Ca²⁺ uptake and the free cytosolic [Ca²⁺] will, however, have to await simultaneous measurements of intracellular Ca²⁺ and SR Ca²⁺ uptake.

Dependency of SR Ca²+ release on temperature. The present data show that the amount of Ca²⁺ released from the SR by a 10-ms depolarization is not affected by a lowering of the bath temperature from 21 to 7°C. In agreement with this, the fast time constant for L-type Ca²⁺ current inactivation, that is modulated by SR Ca²⁺ release (10, 19), was slowed on the first depolarization after clearance of the SR Ca²⁺ content at both 21 and 7°C. Furthermore, the relationship between V_m during depolarization and the Ca²⁺ released from the SR is not altered significantly by the experimental temperature. These results are contrary to data from the mammalian heart where lowering of the experimental temperature has been reported to increase the open probability of the SR Ca²⁺ release channel (2), and a rapid lowering of the bath temperature leads to rapid cooling contractures due to liberation of the SR Ca²⁺ content (2, 3). Assuming that the amount of Ca²⁺ that activates contraction is not very different at the two temperatures, the measured time integrals of the Caf-sensitive tail current after a 10-ms depolarization to +10 mV give a total SR Ca²⁺ release of 1.71 and 1.54 amol/pF at 21 and 7°C, respectively, corresponding to 42 and 38% of the total Ca²⁺ required to activate contraction (see Ref. 10). In this respect, Gillis et al. (6) have reported an increase in the Ca²⁺ sensitivity of troponin C when temperature is lowered, whereas the Ca²⁺ sensitivity of the myofilaments decreases (4), suggesting that there may be temperature-dependent differences in the Ca²⁺ requirements for activation of contraction. One may also assume that the total amount of Ca²⁺ activating contraction equals the sum of the SR Ca²⁺ release plus the total sarcolemmal Ca²⁺ entry (which should be equal to the sarcolemmal Ca²⁺ extrusion obtained in Fig. 4C). This gives a total increase in the cytosolic Ca²⁺ of 4.02 and 3.76 amol/pF (or 61 and 57 µM) at 21 and 7°C, respectively. These values agree well with the passive Ca²⁺ buffering reported in mammals and trout cardiac myocytes at room temperature (8, 10), and they give a relative contribution by the SR of 43 and 41% of the total Ca²⁺ at 21 and 7°C, respectively.

Perspectives