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Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
Submitted 10 September 2002 ; accepted in final form 9 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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sarcoplasmic reticulum; contraction; Ca2+ imaging; fluorescence; excitation-contraction coupling
The spatial and temporal dimensions of sparks are stochastic properties, which vary as probability distributions independent of the initiating stimulus. Sparks typically have a width of 
2 µm, a rise time of 10 ms, and a time constant of decay of 20 ms (1, 4, 8, 28). At the peak, Ca2+ rises to >1.5 times resting levels (4, 12, 29). Decay of spark intensity is believed to represent termination of release flux by ryanodine receptor inactivation (19) combined with diffusion of released Ca2+ away from the site of release and to a lesser extent by uptake into the SR (8). Spontaneous Ca2+ sparks occur at a low rate in quiescent myocytes. However, when a cell is depolarized, it is believed that many sparks are activated in near unison and the combined Ca2+ release constitutes the Ca2+ transient (3, 15, 16, 23).
It is not clear whether the characteristics and behavior of Ca2+ sparks determined in earlier studies apply to mammalian cardiac myocytes at physiological temperature. One study (30) has demonstrated sparks in quiescent rat trabeculae at 33°C. Therefore, sparks can occur above room temperature and are not restricted to dissociated myocytes. However, virtually all other studies that have described and characterized Ca2+ sparks have been conducted in myocytes at room temperature, typically 22–24°C. Changes in temperature can impact on many factors related to cellular Ca2+ dynamics, including SR Ca2+ load, L-type Ca2+ current, duration of contraction, and time course of contraction (1). Interestingly, Sitsapesan et al. (27) have reported that the open probability of the single ryanodine receptor in lipid bilayers is markedly temperature sensitive, with open probability being substantially reduced at higher temperatures. In addition, ryanodine receptors exhibited long open times at low temperature and brief openings at higher temperatures (27). One might speculate that brief open times at physiological temperature might be less likely to result in spontaneous sparks in cardiac myocytes. It is also possible that the characteristics of Ca2+ sparks might be altered by changes in temperature. Therefore, we initiated studies to determine the impact of temperature on the occurrence and characteristics of Ca2+ sparks. The specific objectives of this study were as follows: 1) to determine and compare the incidence and frequency of spontaneous Ca2+ sparks in quiescent mouse ventricular myocytes at 22 and 37°C; 2) to compare the amplitudes, widths, and time courses of sparks at 22 and 37°C; 3) to determine whether changes in sparks with temperature reflect changes in SR Ca2+ stores; and 4) to document changes in Ca2+ transients and contractions occurring with the same changes in temperature in mouse myocytes.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Myocytes released from the pieces of ventricular muscle by gentle swirling were filtered through 225-µm polyethylene mesh (Spectrum). Ca2+ sparks were recorded from myocytes loaded with fluo 3 by incubation in 20 µM fluo 3-AM (Molecular Probes) for 20–25 min at room temperature in the dark. Cells were loaded with dye by adding 200 µl of a fluo 3-AM stock to 800 µl of cell suspension. Fluo 3-AM stock was prepared by dissolving 1 mg of fluo 3-AM in 0.86 ml of anhydrous DMSO (Sigma). To this was added 9 ml of a solution containing 300 µl of pluronic F-127 (Molecular Probes) dissolved and sonicated in 12 ml of fetal calf serum. Myocytes loaded with fluo 3 were transferred to an experimental chamber (volume: 1.5 ml) mounted on the stage of a Zeiss LSM 510 laser scanning microscope (Axiovert 100). Cells were allowed to settle (5–10 min) and adhere to the glass bottom of the chamber, which was formed by a glass coverslip (24 x 50 mm, 0.08–0.13 mm in thickness, VWR Scientific). Coverslips were coated with poly-L-lysine (Sigma) at a concentration of 5–10 µg/cm2. Surfaces treated with poly-L-lysine were air dried and washed with distilled water before use. After myocytes had adhered, they were superfused at 1.3 ml/min at either room temperature (22–24°C) or 37°C with a solution of the following composition (in mM): 145 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). The temperature of the superfusate was controlled by a water-jacketed heat exchanger, in which the physiological buffer passes through stainless steel tubing to maximize heat exchange. The heat exchanger was positioned immediately before the bath inlet. The temperature gradient across the bath was <1°C. Effects of the temperature gradient were minimized by always selecting cells from the middle of the bath.
Experiments were conducted on quiescent myocytes or on myocytes stimulated through bipolar silver wire electrodes positioned for field stimulation. Stimuli were rectangular pulses, 5 ms in duration, delivered at 1 Hz (model SD9, Grass Instruments). The stimulus voltage was adjusted to 25% higher than the value required to initiate visible contractions.
Changes in free Ca2+ were measured in line scan mode. Fluo 3 was excited at 488 nm, and emission intensity was measured at 525 nm (Zeiss oil immersion objective, x40/1.3 numerical aperture). Myocytes were repetitively scanned along the entire length of the cell at 1.5-ms intervals, for a maximum of 6 s. The confocal pinhole was adjusted to 54 µm to provide a maximum x-y-z resolution of 0.26 x 0.26 x 0.75 µm, and each line was composed of 512 pixels (voxels). The laser intensity was reduced to 
5% maximum to decrease cell damage and dye bleaching. Line scan diagrams were constructed by stacking emission lines, corresponding to excitation scans, in temporal order.
Images were analyzed with Scion Image (Scion), ImageJ (NIH), and SigmaPlot (version 5.0, Jandel Scientific). Ca2+ sparks were identified as local peak elevations of fluorescent intensity (F) that were 
1.5 times the surrounding background levels (F0). The parameters that were measured included amplitude (F/F0), spatial width [full width at half-maximum intensity (FWHM)], time to peak (TTP) amplitude, and time for decay to half-peak intensity (T
). In addition, the frequency of sparks (number of sparks·100 µm–1·s–1) and the incidence (%) of myocytes exhibiting sparks during the 6-s recording period were measured. In field-stimulated myocytes, Ca2+ transients were determined by averaging the intensity of each sequential scan line and plotting the mean intensity as a function of time. These Ca2+ transients represent the average change in Ca2+ along the length of the cell but are restricted to the volume excited by the laser scan. TTP, T, and F/F0 were measured for Ca2+ transients recorded at both temperatures. Fluorescence emission was not corrected for temperature, because spark amplitude was measured as the ratio of the peak fluorescence to adjacent background diastolic levels. Because changes to peak and background fluorescence are expected to be proportional, the ratio should be unaffected. This was confirmed in vitro for the same optical path and laser intensity by determining the ratio of emission recorded for 300 and 150 nM free Ca2+ at both 22 and 37°C. The emission ratio was identical at both temperatures. Because the ratio of F to F0 was not affected by temperature, the remaining spark parameters as well as our criterion for spark detection should not be affected by temperature.
In additional experiments, Ca2+ concentrations were measured ratiometrically with fura 2 with a Photon Technology International DeltaRAM system and Felix software (PTI; Brunswick, NJ) as described previously (17, 31). Fura 2-AM stock solution (2.5 mM) was prepared in anhydrous DMSO. Cells were incubated in 5 µM fura 2-AM for 20–30 min at room temperature in the dark. Myocytes were transferred to an experimental chamber mounted on the stage of a Nikon inverted microscope (Eclipse TE200) and field stimulated at 1 Hz. Cells loaded with fura 2 were excited alternately at 340 and 380 nm, and emission was recorded at 510 nm. Light collection was restricted to the cell by an adjustable aperture. Background fluorescence was subtracted for each excitation wavelength, and the ratio of emission during excitation at 340 and 380 nm was converted to Ca2+ concentration with calibration curves determined in vitro with the same optical path. To compensate for changes in fluorescence with temperature, calibration curves were determined at both 22 and 37°C. Free Ca2+ concentrations were buffered by EGTA in the calibrating solution (1 µM fura 2, 10 mM EGTA, 100 mM KCl, and 10 mM K-MOPS). pH was adjusted to neutrality with KOH (30 mM) at both temperatures, and free Ca2+ concentrations in the calibrating solutions were calculated for each temperature with Maxchelator (version 2.40, Win-MAXC). Unloaded cell shortening was measured with a video edge detector (Crescent Electronics; Sandy, UT) coupled to a closed circuit television system (31).
In some experiments, SR Ca2+ stores were assessed by rapid application of 10 mM caffeine with a temperature-controlled rapid solution switcher (11). For this purpose, caffeine was dissolved in solution with 0 mM Na+ and 0 mM Ca2+ to prevent efflux of Ca2+ through Na+/Ca2+ exchange (13). The composition of this solution was (in mM) 140 LiCl, 4 KCl, 4 MgCl2, 5 HEPES, 10 glucose, 0.3 lidocaine, and 4 4-aminopyridine (pH 7.4 with LiOH). Released Ca2+ was measured by the 340-to-380-nm fura 2 ratio as described above. Cells in these experiments were activated regularly with 200-ms voltage-clamp pulses from a holding potential of –80 to 0 mV. Caffeine was applied for 1 s after a train of five conditioning pulses.
Statistical analyses were conducted with SigmaStat (Jandel Scientific). Differences between means were tested for significance with either a t-test or a rank sum test. Differences in incidence were subjected to a 
2-test. Differences were considered statistically significant for P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The line scan diagrams in Fig. 1, A and B, show a difference in the number of sparks occurring in resting cells at different temperatures. We evaluated the effect of temperature on the number of cells exhibiting sparks during a 6-s line scan. Figure 1C shows that close to 90% of cells exhibited Ca2+ sparks at 22°C, whereas only 20% of myocytes generated Ca2+ sparks when the temperature was 37°C. The percentage of cells exhibiting sparks was significantly higher at 22°C compared with 37°C. We also determined the overall frequency of sparks occurring at the two different temperatures. Figure 1D shows that the frequency of Ca2+ sparks was markedly and significantly decreased at 37°C compared with 22°C.
We next determined and compared the characteristics of Ca2+ sparks recorded at both temperatures. Figure 2, A and B, shows line scan diagrams of representative Ca2+ sparks recorded at 22 and 37°C, respectively. The same sparks are shown rotated and with intensity axes added in the vertical plane in Fig. 2, C and D. The example recorded at 22°C appears somewhat slower in time course than the example recorded at 37°C. Intensity-time profiles for the same sparks are presented in Fig. 2, E and F. Here, the relative intensity is presented as the ratio of the fluorescence intensity at any given point (F) to the background intensity preceding the spark (F0). The spatial contours of the same sparks are shown by the relative intensity-distance profiles in Fig. 2, G and H. The widths of these representative Ca2+ sparks appeared to be similar at the two temperatures.
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Quantitative data for Ca2+ spark characteristics were derived through measurements illustrated in Fig. 3A. Figure 3A shows a surface plot of fluorescence intensity during a Ca2+ spark. Time and distance are plotted on the x- and y-axes, and intensity is plotted on the z-axis. The amplitudes of sparks were determined as F/F0 at the peak of the spark. Mean data for amplitudes of sparks at 22 and 37°C are shown in Fig. 3B. Spark amplitudes were significantly lower at 37°C compared with amplitudes measured at 22°C. Spatial widths of sparks were measured as FWHM, as indicated in Fig. 3A. Figure 3C shows that FWHM was virtually identical at the two temperatures tested. Time courses of Ca2+ sparks were divided into rising and decaying phases. The rising phase was measured as TTP, and the decay phase was measured as T, as indicated in Fig. 3A. Figure 3D shows that TTP was significantly shorter for Ca2+ sparks recorded at 37°C. In contrast, there were no significant differences between T for sparks at 22 and 37°C.
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The frequency distributions of spark characteristics at both temperatures are plotted in Fig. 4. Figure 4A shows the distribution of spark amplitudes. The amplitude with the greatest number of events was the same at both temperatures. However, there was a greater number of large sparks at 22°C. Thus the frequency distribution for amplitudes was broader at 22°C compared with 37°C. The frequency distributions for FWHM were similar at both temperatures (Fig. 4B). However, the frequency distributions for TTP indicate that the distribution of rise times was shifted to shorter times at 37°C (Fig. 4C). The TTP with the greatest number of events also was shorter at 37°C. The distribution frequencies for T were similar for both temperatures (Fig. 4D).
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Because Ca2+ transients are believed to represent the sum of numerous Ca2+ sparks, we determined whether changes in temperature also resulted in changes in Ca2+ transients in field-stimulated myocytes. Figure 5A shows a representative line scan diagram recorded at 22°C. Three stimulated Ca2+ transients were triggered during this line scan. To visualize the time courses of the large Ca2+ transients, fluorescent intensity was averaged for each line in the diagram and plotted as a function of time below the line scan diagram to obtain a spatially averaged transient. A representative line scan diagram recorded at 37°C plus the corresponding spatially averaged Ca2+ transients below are shown in Fig. 5B. The time courses of the Ca2+ transients recorded at the two temperatures were similar. In both line scans shown in Fig. 5, the scan lines extended slightly beyond both ends of the cells. Therefore, the contractions of the cells are visible as scalloping of the edges of the fluorescent signal. The contractions at 37°C appeared to be more rapid than the contractions at 22°C. In addition to the large Ca2+ transients, several Ca2+ sparks were observed between transients at both temperatures.
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Mean data for amplitudes and time courses of Ca2+ transients recorded at both temperatures are summarized in Fig. 5, C–E. Figure 5C shows that there was no difference between the mean amplitudes of Ca2+ transients expressed as F/F0. Mean TTP is shown in Fig. 5D. Although there was a trend toward shorter TTP at 37°C, this difference was not statistically significant. Figure 5E shows that T was similar at both temperatures. There also was no significant difference in the number of cells exhibiting Ca2+ sparks between stimulated transients. At 22°C, 42% of 31 cells exhibited Ca2+ sparks, and at 37°C, 47% of 19 cells exhibited sparks.
Spatially averaged transients determined with fluo 3 provide a measure of changes in free intracellular Ca2+ relative to resting levels (F0) but do not provide a measure of actual Ca2+ concentrations. To determine whether Ca2+ concentrations achieved during transients were different at 22 and 37°C, we conducted experiments with similar field stimulation protocols in cells loaded with the ratiometric dye fura 2. Unloaded cell shortening was measured simultaneously. Figure 6, A and B, shows representative recordings of transients and contractions measured at 22 and 37°C, respectively. Mean data for amplitudes and time courses of transients and contractions are shown in Fig. 6, C–F. Figure 6C presents mean concentrations of Ca2+ for transients recorded at both temperatures. The diastolic Ca2+ concentration, measured immediately before the beginning of transients, was not significantly different at 37°C compared with 22°C. The peak systolic Ca2+ concentration and the amplitudes of Ca2+ transients also were not significantly different at 37°C compared with 22°C. The mean amplitudes of contractions were slightly larger at 37°C, but this difference was not statistically significant (Fig. 6D). Mean values for TTP and T of the Ca2+ transients are shown in Fig. 6E. TTP and T were significantly faster at 37°C. Similar significant changes in the time courses of contractions were observed, as shown in Fig. 6F. The abbreviation of time courses was greater for contractions than observed for transients. However, this difference may reflect in part the kinetics of fura 2.
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The differences in incidence and frequency of spontaneous sparks in resting myocytes at 22 and 37°C could reflect a change in SR Ca2+ content (24, 25). Therefore, we assessed the SR Ca2+ content with rapid applications of 10 mM caffeine in cells loaded with fura 2. To stabilize the myocytes during rapid solution switches, the myocytes were impaled with high-resistance electrodes. Caffeine was rapidly applied for 1 s after the last of five conditioning pulses. The caffeine was delivered in buffer with 0 mM Na+ and 0 mM Ca2+ to prevent efflux of Ca2+ through the Na+/Ca2+ exchanger. Thus the peak amplitude of the caffeine-induced transient is a measure of caffeine-releasable SR stores (13). Figure 7, A and B, shows recordings of Ca2+ concentrations in representative myocytes at 22 and 37°C, respectively. Each trace shows the resting Ca2+ concentration followed by Ca2+ transients induced by five conditioning pulses. In Fig. 7, A and B, the train of small transients is followed by a large, long-lasting transient induced by application of caffeine. The peak amplitude of the caffeine-induced transient was smaller at 37 than at 22°C. Figure 7C shows mean data for resting Ca2+ concentration and peak caffeine-induced transients. The mean Ca2+ concentration at rest was similar at 37 and 22°C. However, the mean peak Ca2+ concentration elicited by caffeine was significantly decreased at 37°C compared with 22°C. These data indicate that SR Ca2+ content decreased by 32% when the temperature was increased to 37°C.
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We next investigated whether changes in Ca2+ sparks observed with increased temperature were caused by decreased SR Ca2+. To do this, we determined the Ca2+ spark frequency at 37°C in myocytes in which SR Ca2+ stores were increased by superfusion with extracellular solution containing 5 mM Ca2+. SR Ca2+ stores were assessed in cells loaded with fura 2 by rapid application of 10 mM caffeine in 0 mM Na+ and 0 mM Ca2+. Figure 8A shows representative caffeine-induced transients recorded at 37°C in the presence of 1 and 5 mM extracellular Ca2+. Figure 8B presents mean data that shows that superfusion with 5 mM Ca2+ significantly elevated SR Ca2+ stores. The relative increase in SR stores with 5 mM Ca2+ closely approximated the relative decrease caused by increasing temperature. We then used the same increase in extracellular Ca2+ concentration to assess whether increasing SR Ca2+ would counteract the effects of temperature on spark frequency and parameters in cells loaded with fluo 3. Figure 8C compares the frequency of Ca2+ sparks measured at 22 and 37°C in the presence of 1 mM Ca2+ and at 37°C in the presence of 5 mM Ca2+. Ca2+ spark frequency decreased significantly when the temperature was increased with 1 mM Ca2+. However, when cells were exposed to 5 mM Ca2+ at 37°C, spark frequency was no longer significantly decreased, although spark frequency did not return fully to control. These data demonstrate that changes in SR Ca2+ stores likely account, at least in part, for the effect of temperature on Ca2+ spark frequency.
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It is possible that the changes in Ca2+ spark parameters observed with increasing temperature also might be reversed by increasing SR Ca2+ stores. Therefore, we compared Ca2+ spark parameters recorded in 1 and 5 mM extracellular Ca2+ at 37°C. Figure 9A shows that F/F0 did not increase when cells were exposed to 5 mM Ca2+. Similarly, spark width, TTP, and T were not affected significantly by increasing extracellular Ca2+ to 5 mM (Fig. 9, B–D). Thus increasing SR Ca2+ stores by exposing myocytes to elevated extracellular Ca2+ did not reverse the effects of warming on Ca2+ spark parameters.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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of sparks with temperature. These changes in occurrence and characteristics of sparks were accompanied by a decrease in SR Ca2+ stores at 37°C. When SR Ca2+ stores were increased to compensate for this effect of temperature, spark frequency was no longer decreased, but changes in spark parameters were not eliminated. Changes in Ca2+ sparks with increased temperature were not accompanied by changes in the amplitudes of Ca2+ transients measured with fluo 3 or fura 2 in field-stimulated cells. However, TTP and half-relaxation of fura 2 transients and contractions were significantly faster at 37°C, although fluo 3 spatially averaged transients did not exhibit changes in time course. One of the key observations in this study is that the occurrence of spontaneous Ca2+ sparks is greatly decreased at 37°C in mammalian ventricular myocytes. Thus observations of spark frequency and incidence at room temperature are not a good measure of their occurrence at physiological temperature. Several possible mechanisms that might explain why the occurrence of sparks decreases at physiological temperature should be considered. First, it is unlikely that the temperature dependence of Ca2+ sparks reflects changes in open probability of L-type Ca2+ channels, as spontaneous sparks are not believed to be triggered by opening of L-type Ca2+ channels (25, 29). Second, it has been reported that Ca2+ spark frequency increases with increases in cytosolic Ca2+ concentration (1). However, in the present study, we did not observe significant changes in diastolic Ca2+ when temperature was increased to 37°C. Third, SR Ca2+ stores might play an important role in determining Ca2+ spark frequency. Several studies (18, 24, 25) have reported an increase in spark frequency when SR Ca2+ increases. In some mammalian species, such as the rabbit and ferret, SR Ca2+ stores are greater at low temperatures compared with higher temperatures (22). However, in other species, such as the cat, SR Ca2+ stores do not change significantly between 25 and 35°C (22). When we measured SR Ca2+ stores in mouse ventricular myocytes with caffeine applications and fura 2, we found that there was a significant decrease at 37°C compared with 22°C. Interestingly, when SR Ca2+ stores were increased at 37°C, a significant decrease in spark frequency was no longer observed. This suggests that changes in SR Ca2+ are likely to contribute to the decrease in spark incidence and frequency observed at 37°C in mouse myocytes.
It also is possible that temperature alters ryanodine receptor function. Increased temperature decreases the open probability and open times of single sheep ryanodine receptors in lipid bilayers (27). If similar changes occur in vivo in mouse myocytes, this may provide another mechanism that contributes to the decreased incidence and frequency of Ca2+ sparks at physiological temperature. Decreased open probability of ryanodine receptors at 37°C compared with 22°C might decrease the likelihood that any given opening of a ryanodine receptor would recruit neighboring ryanodine receptors and initiate a spark. The decreased open times reported by Sitsapesan et al. (27) also might explain the reduced TTP and decreased amplitude of Ca2+ sparks observed at 37°C in the present study. If ryanodine receptors close sooner at physiological temperature and thereby curtail Ca2+ release, both a decreased TTP and decreased spark amplitude would be expected. Interestingly, the effects of temperature on TTP and amplitude of sparks were not reversed by increasing SR Ca2+ stores. This suggests that these effects of temperature are not mediated by changes in SR stores and therefore may reflect changes in ryanodine receptor kinetics. This interpretation is supported by observations by Lukyanenko et al. (18), who investigated the effects of changing SR stores at room temperature. They found that Ca2+ spark frequency paralleled SR stores; however, spark amplitudes were unchanged.
We also found that neither spatial width nor T of sparks changed with temperature. The absence of a change in spark width suggests that the number of ryanodine receptors comprising a spark unit was the same at both 22 and 37°C. The absence of a significant change in T between 22 and 37°C in the present study indicates that decay of sparks occurs by a mechanism that is not strongly affected by temperature. Indeed, T is believed to be determined primarily by diffusion of Ca2+ away from the release sites, whereas reuptake of Ca2+ by the SR plays a lesser role (8). Our results are in keeping with diffusion being the main determinant of spark decay, as the Q10 for aqueous diffusion is only 1.3 (10).
The changes observed in spontaneous Ca2+ spark amplitude did not correlate with changes in the amplitudes of whole cell transients determined with either fura 2 or fluo 3. Spark amplitudes decreased, whereas the amplitudes of Ca2+ transients were unchanged. Thus the change in unitary release of SR Ca2+ observed with spontaneous sparks did not impact on the amplitudes of Ca2+ transients. However, Ca2+ transients are not caused by spontaneous sparks, and it is possible that depolarization-induced sparks respond differently to temperature than spontaneous sparks. Alternatively, an increase in number of spark units recruited by depolarization at 37°C might compensate for reduced spark amplitudes.
The TTP for whole cell Ca2+ transients measured with fura 2 was shorter at 37°C compared with 22°C. A trend toward shorter TTP was also observed with spatially averaged fluo 3 transients, although this was not statistically significant. It is not clear whether abbreviation of rise times of Ca2+ transients reflects the stochastic properties of Ca2+ sparks. Other factors such as synchrony of spark recruitment and the length of the latent period between depolarization and initiation of sparks may play a significant role in determining the TTP. For example, slowed rise times of Ca2+ transients observed in myocytes from failed hearts have been associated with a greater dispersion in latency for spark initiation (14). Furthermore, synchronization of sparks and shortening of latent periods with isoproterenol resulted in shorter rise times for transients (14). It is unknown whether increasing temperature causes a similar synchronization and decreased latency in depolarization-induced Ca2+ sparks. However, if such a change occurs, it might explain why the peak amplitude of Ca2+ transients was preserved despite a reduction of SR Ca2+ stores at 37°C. In addition, the magnitude of trigger Ca2+ current is increased substantially at 37°C. L-type Ca2+ current exhibits a Q10 of 3, which is believed to reflect in part an increase in phosphorylation levels and therefore open probability (20). L-type Ca2+ current also may make a larger contribution to the transient and thereby help preserve the amplitudes of the transients despite the reduction in SR Ca2+ stores.
The T of Ca2+ transients measured with fura 2 decreased significantly at 37°C compared with 22°C. In contrast, there was no significant change in T of spatially averaged Ca2+ transients detected with fluo 3. The rate of dissociation of Ca2+ from fura 2 is much slower than that from fluo 3 (7). Thus the decay observed in the fura 2 transients may largely represent the slow kinetics of Ca2+ dissociation from fura 2. Because of this, the marked change in decay time of Ca2+ transients detected with fura 2 may reflect the substantial temperature dependence of the fura 2 dissociation constant (9). The dissociation constants for fluo 3 are much higher than those for fura 2 at both room and physiological temperatures (7), and therefore the time courses of fluo 3 transients more likely reflect changes in free Ca2+. Therefore, our data suggest that there is little change in T of Ca2+ transients at 37°C compared with 22°C in mouse ventricular myocytes.
Although the time course of decay of fluo 3 Ca2+ transients changed little with temperature, times to half-relaxation of contractions decreased at 37°C compared with 22°C. It is not clear what causes this disparity in time courses. It is possible that additional temperature-sensitive events involved in contraction and relaxation may be affected differently from events related to SR Ca2+ release. For example, rates of Ca2+ dissociation from myofilaments as well as steps in hydrolysis of ATP and myofilament movement may vary substantially at different temperatures. Thus the relationship between declining Ca2+ concentrations and relaxation might differ at 22 and 37°C.
The present study demonstrates that the stochastic properties as well as the frequency and incidence of spontaneous Ca2+ sparks recorded in isolated mouse myocytes at room temperature do not accurately reflect spark characteristics at physiological temperature. This finding has important implications regarding our understanding of the dynamic exchange of Ca2+ between intracellular and extracellular compartments. The level of SR Ca2+ reflects a balance between uptake and release. Spontaneous Ca2+ sparks provide a mechanism for spontaneous SR Ca2+ leak. The present study suggests that SR Ca2+ leak in the form of sparks is much less at physiological temperature than at room temperature, as frequency, rise times, and amplitudes of sparks are decreased. Changes in SR Ca2+ stores may account in part for the effect of temperature on spark frequency, although other factors such as changes in the open probability of ryanodine receptors also may contribute.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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