Behavior of Ca2+ waves in multicellular preparations from guinea pig ventricle

Nagomi Kurebayashi,1 Haruyo Yamashita,2 Yuji Nakazato,2 Hiroyuki Daida,2 and Yasuo Ogawa1

Departments of 1Pharmacology and 2Cardiology, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Submitted 26 April 2004 ; accepted in final form 7 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ca+ waves have been implicated in Ca2+ overload-induced cardiac arrhythmias. To deepen understanding of the behavior of Ca2+ waves in a multicellular system, consecutive two-dimensional Ca2+ images were obtained with a confocal microscope from surface cells of guinea pig ventricular papillary muscles loaded with fluo 3 or rhod 2. In intact muscles, no Ca2+ waves were detected under the resting condition, whereas they were frequently observed during the rest immediately after high-frequency stimulations where cytoplasmic Ca2+ concentration and Ca2+ stored in the sarcoplasmic reticulum (SR) were gradually decreasing. The intervals of Ca2+ waves increased as they occurred later, their amplitudes and velocities remaining unchanged. A SERCA inhibitor reversibly prolonged the wave intervals. In Na+-free/Ca2+-free medium where neither Ca2+ influx nor Na+/Ca2+ exchange took place, recurrent Ca2+ waves emerged at constant intervals in each cell. These results are consistent with the conclusion that the loading level of the SR is critical for induction of Ca2+ waves. Each cell independently exhibited its own regular rhythm of Ca2+ wave with a distinct interval. These waves propagated in either direction along the longitudinal axis within a muscle cell, but seldom beyond the cell boundary. In contrast, in partially damaged muscles that showed spontaneous Ca2+ waves at rest in normal Krebs solution, their propagation often was unidirectional, decreasing in frequency. In these cases, however, Ca2+ waves rarely moved beyond the cellular boundary. The gradient of the cytoplasmic Ca2+ concentration was suggested to be the cause of the one-way propagation.

wave propagation; luminal calcium ion; cytoplasmic calcium ion; sodium/calcium exchange


IN EXCITATION-CONTRACTION coupling in mammalian cardiac muscle, Ca2+ influx through activated dihydropyridine receptors triggers the opening of ryanodine receptors in the sarcoplasmic reticulum (SR) of muscle cells, resulting in a transient elevation of cytosolic Ca2+ sufficient for muscular contraction. In recent years, the development of confocal fluorescence microscopy with high spatial resolution has led to the identification of "Ca2+ sparks," spontaneously or electrically triggered and localized Ca2+ release events from SR (4, 20). Ca2+ sparks are believed to be "elementary events" of cardiac excitation-contraction coupling in the sense that whole cell calcium release can be plausibly reconstructed as a summation of these events. In the Ca2+-overloaded condition, however, Ca2+ is spontaneously released from SR and propagates throughout the cell in a wave, the "Ca2+ wave." The Ca2+ wave may lead to aftercontraction and delayed afterdepolarization (DAD; see Refs. 5 and 24), which is probably caused by enhanced extrusion of Ca2+ via the Na+/Ca2+ exchange reaction. The elevated Ca2+ might also activate Cl efflux through Ca2+-activated Cl channels (31, 35). The DAD can trigger an extrasystolic action potential when it reaches the threshold. Thus Ca2+ waves could be a candidate for the mechanism underlying cardiac arrhythmia (28).

Ca2+ waves have been studied extensively, primarily with enzymatically isolated single cardiac cells (6, 19, 28), and have been shown able to induce action potential (5, 24). Those preparations, however, lack cell-to-cell interactions through gap junctions, which have critical roles in the conduction of cellular excitation and generation of arrhythmia. To date, only a limited number of groups have investigated Ca2+ waves in multicellular cardiac muscle preparations (1, 13, 18, 23, 26, 33). Wier and colleagues (33) injected fluo 3 in rat trabeculae and showed that Ca2+ waves did not propagate beyond cells (33). Kaneko et al. (13) monitored Ca2+ waves on the outer surface of cardiac cells in Langendorff-perfused rat heart. They classified waves into three different categories on the basis of resting fluorescence intensity, which probably reflected viability of the cells, and concluded that Ca2+-overloaded waves showed a high probability of propagation whereas the other types of Ca2+ waves were less likely to propagate. These reports, although informative, were carried out mostly with rat heart muscles. It is well known that there are marked differences among animal species in Ca2+ homeostasis in heart muscle. Contribution of Ca2+ influx and SR Ca2+ release to the Ca2+ transients differs in magnitude among animals; SR Ca2+ release is the greatest in rat and mouse ventricular muscles, among those including guinea pig and human hearts (3). Other properties specific to rat heart are also reported (3). This makes results with hearts from animals other than rat of great interest.

It is interesting to know whether Ca2+ waves propagate across the cell boundary, because if so the possibility of spontaneous activity/DAD would be greatly increased by the intercellular propagation. In this study, we took real-time images of Ca2+ waves in intact ventricular papillary muscles from guinea pigs to examine whether cell-to-cell propagation of Ca2+ waves could be observed and how they behaved in multicellular tissue. We monitored Ca2+ waves induced by high-frequency electrical stimulation, which caused temporal Ca2+ overload and often an aftercontraction. In addition, Ca2+ waves in damaged muscles, where they occurred spontaneously, were also monitored. Our results indicate that Ca2+ waves seldom propagated across the cell boundary, although they often appeared to flow in a fixed direction. Preliminary results have been published (15).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation. All experiments were carried out in accordance with Juntendo University Ethics Committee guidelines. Guinea pigs were anesthetized with an intraperitoneal injection of pentobarbital sodium. The heart was removed quickly from the chest and then perfused via the aorta with high-K+ (25 mM KCl added) Krebs solution. Papillary muscle bundles (0.5–1.5 mm in diameter, ~3 mm in length) were excised from left and right ventricles after the muscle tendon and basal portion of the bundle had been secured with silk thread in the high-K+ solution.

Confocal imaging of Ca2+ waves. The muscle bundles were incubated with a Ca2+ indicator (fluo 3-AM or rhod 2-AM) at 10 µM for 80 min at room temperature in normal Krebs solution. After the dye was washed out, the muscle bundle was connected to hooks in a chamber with thread (14). The chamber, the bottom of which was made of a coverslip, was placed on the stage of an inverted microscope. The bundle was stretched to ~110% of its slack length and gently pushed toward the bottom with a Plexiglas plate so that the lower surface of the bundle was several micrometers above the bottom. Through this gap, cells at the lower surface were allowed access to the bathing solution. The chamber was perfused with Krebs solution at a rate of 2 ml/min. Experiments were carried out at 25–27°C.

The bundles were viewed with a confocal laser scanning microscope system (Oz system; Noran Instruments) equipped with an Argon Krypton Ion Laser System (488 and 568 nm excitation). Fluo 3 was excited at 488 nm, and fluorescence was measured at wavelengths of >500 nm, whereas rhod 2 was excited at 568 nm and fluorescence of >590 nm was detected. To observe multiple cells on the bundle, a x10 or x20 objective lens was used. The z-axis resolution as estimated by imaging fluorescent beads (Noran Instruments) was 5 and 15 µm for the x20 and x10 objective lens, respectively. Ca2+ images sometimes included waves from cells at the second layer when the focused plane was close to the interface of the stacked cells. However, even in such cases, we were able to identify overlapping Ca2+ waves from the second layer cells because their fluorescence intensity was much lower than those of the surface layer cells.

Intact muscle preparations were usually conditioned by stimulating at 0.5 Hz for >5 min in normal Krebs solution by a pair of platinum-plate electrodes (25 mm in length) using rectangular current pulses (1 ms, 1.5 threshold voltage). For induction of waves in an intact muscle preparation, the muscle was stimulated at a higher frequency, 2~3 Hz, for 3~5 min, and then the stimulation was stopped. Because Ca2+ waves occurred after the train of stimulation, acquisitions of fluorescent images were started 3–4 s before the cessation of a series of stimulations. In typical experiments, each image was taken with 256 x 240 pixels every 8.3 ms, and eight images were averaged to get a single image; this averaged image was obtained every 67 ms. These 500 averaged images were taken continuously for one measurement of 33 s. During the 33-s measurements, fluorescence intensity of fluo 3 decreased by ~10% because of quenching, as determined from peak fluorescence intensity at 0.5 Hz stimulation. In addition, fluorescence intensity gradually decreased with time without any illumination of laser light, probably because dye was diffusing through the gap junction from surface cells to those at deeper regions. The change in intensity was 10~20%/h.

Damaged muscles, which showed spontaneous and recurrent Ca2+ waves for a long period (>15 min) at rest, were obtained by either of the following treatments. 1) A muscle was cut at one end (mechanical injury), and cells near the cut end then showed spontaneous Ca2+ waves. 2) A muscle was stimulated at 2 Hz for ~30 min without perfusing Krebs solution (overload and anoxia). 3) A muscle was first incubated in Ca2+-free Krebs solution for ~30 min and then challenged with normal Krebs solution (Ca2+ paradox). Data were obtained 15~45 min after these treatments. In a specimen subjected to one of these treatments, damaged cells showed varied viability with different magnitudes of impairment.

In this study, we did not use any reagents to immobilize muscle bundles. The muscle bundle moved quite a bit in response to electrical stimulation and often moved out of the field of view and/or out of focus. Therefore, the Ca2+ transient signals on electrical stimulation suffered from considerable movement artifacts. However, Ca2+ waves that occurred after the series of stimulations were analyzable because the movements caused by the Ca2+ waves, if any, did not affect the results.

Solutions. Normal Krebs solution contained (in mM): 120 NaCl, 5 KCl, 25 NaHCO3, 1 NaH2PO4, 2 CaCl2, 1 MgCl2, and 10 glucose. For low-Na+ (26 mM Na+) solutions, NaCl was replaced with the equimolar LiCl; these solutions were saturated with 95% O2-5% CO2. In some experiments, a HEPES-buffered solution was used instead (in mM): 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES, pH adjusted to 7.4 with NaOH. For a Na+-free/Ca2+-free (0Na+/0Ca2+) solution, CaCl2 was omitted, NaCl was replaced with equimolar LiCl, and pH was adjusted to 7.4 with LiOH. Each HEPES-buffered solution was saturated with 100% O2. When the effects of acidic pH were determined (the last part of RESULTS), a bicarbonate-buffered Na+-free/Ca2+-free (bicarbonate-buffered 0Na+/0Ca2+) solution was used in which all Na+ in normal Krebs solution was replaced with equimolar Li+ and CaCl2 was omitted. This solution was saturated either with 95% O2-5% CO2 (pH 7.4) or 70% O2-30% CO2 (pH 6.7). Cyclopiazonic acid (CPA) was dissolved in DMSO and added to modified Krebs solutions at a final DMSO concentration of 0.1%.

Fluo 3-AM and rhod 2-AM were obtained from Dojindo (Kumamoto, Japan) and Molecular Probes (Eugene, OR), respectively. The Ca2+ signals from fluo 3 and rhod 2 showed the same time course of Ca2+ transient in double-stained muscle cells (data not shown), although Fmax/F0 values were somewhat different. Therefore, all the data of Ca2+ waves were analyzed similarly.

Data analysis. A series of the image data obtained with the Noran Oz system was converted to stacks of 500 TIFF images for analysis by National Institutes of Health Image software. Time-based scan images were obtained along lines drawn on the stacks of images with "Reslice" command and were used to determine wave amplitudes, velocities, and intervals. All averaged values were expressed as means ± SD. Statistical analysis was performed using ANOVA for multiple comparisons and Student's t-test. P values <0.05 were considered to be significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In guinea pig papillary muscle, intact muscle cells showed low background fluorescence and no obvious change at rest, but responded to stimulation with a marked increase in fluorescence intensity as Ca2+ transient. Most results in this study were initially obtained with these intact preparations to characterize the Ca2+ waves. Later experiments with damaged cells were performed to understand the pathological implications.

Figure 1 shows typical experiments. The specimen was first stimulated for 5 min at 1 Hz and then kept unstimulated for >30 s (A–C). A series of two-dimensional Ca2+ images of fluorescence changes at the last 2~3 s of stimulation and ~30 s of unstimulated periods was acquired for each determination, as described in MATERIALS AND METHODS. During 1 Hz stimulation, the muscle cells showed an increase in Ca2+ signal in response to each electrical stimulation, and, after the cessation of stimulation, the fluorescence intensities gradually and monotonically decreased without Ca2+ waves (Fig. 1B). The fluorescence signal taken from a wider field of view (Fig. 1A) that contained ~20 cells also showed a monotonic decrease (Fig. 1C). Similar determinations with the initial 2 Hz stimulation followed in succession (Fig. 1, D–F). Stimulation at 2 Hz also caused fluorescent Ca2+ transient in individual cells (Fig. 1E). A remarkable difference, however, was observed after the cessation of electrical stimulation. After 2 Hz stimulation, Ca2+ waves or Ca2+ oscillations were intermittently observed (Fig. 1, D and E; please refer to the Supplemental Material for this article to view renderings).1 A time course of the Ca2+ signal taken from the entire field of view that corresponds to an ensemble signal from ~20 cells showed a hump overlaying a gradual decrease in the Ca2+ concentration after cessation of electrical stimulation, resembling a profile of "aftercontraction" (Fig. 1F). The following descriptions were focused on the Ca2+ waves during the period of nonstimulation after the train of stimulations at a specified frequency.



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Fig. 1. Induction of Ca2+ waves after high-frequency stimulation in a papillary muscle. The muscle, loaded with fluo 3, was stimulated at 1 Hz (A–C) or 2 Hz (D–F) for 5 min, and then stimulation was stopped. See MATERIALS AND METHODS for details. Images taken 7.6 s after cessation of stimulation are shown in A and D. Time courses of fluorescence changes from the middle region of four cells indicated by small colored squares are plotted in B and E. C and F show time courses of fluorescence intensities (F. I.) taken from the large white squares. Bright white granules in A and D are endocardial endothelial cells, which did not respond to electrical stimulation. The muscle fiber was observed with a x10 objective. Please refer to the Supplemental Material for this article (published online at the American Journal of Physiology-Cell Physiology web site) to view a video for D.

 
To analyze Ca2+ waves more systematically, we obtained time-based scan images from the already-acquired stack of images (see MATERIALS AND METHODS). A time-based scan image taken along a broken line drawn on the four cells (a–d) contoured in Fig. 2A is shown in Fig. 2B. The horizontal axis of Fig. 2B corresponds to the location along the broken line as shown at the top of Fig. 2B, whereas the vertical axis corresponds to time during image sampling. Fluorescence transients of the four cells synchronously increased during field stimulation. During the following period of nonstimulation, however, the increase in fluorescence was asynchronous. On closer examination, those fluorescence increases occurred at localized points in each cell, then propagated within the cell in the Ca2+ wave, and finally stopped at the cell boundary. The origins of the waves were not necessarily fixed within the individual cells although some appeared to have fixed origins. In some cells, Ca2+ waves originated at multiple points as reported on rat cardiac muscle (2) (see the third wave in cell b of Fig. 2B). Direction of intracellular propagation of Ca2+ waves along the longitudinal axis was variable among cells (bidirectional) under this condition.



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Fig. 2. Ca2+ waves in multicellular cardiac preparation. A: image of the fluo 3-loaded papillary muscles. Regions enclosed by white lines are contours of 4 cells aligned transversely. B: time-based scan image obtained along a broken line drawn on the 4 cells in A. Note that all Ca2+ waves stopped at the cell boundary. C: time-based scan images of 4 consecutive Ca2+ waves with expanded time scale, which were taken from B, cell b. Nos. 1–4 indicate the first to the fourth Ca2+ waves. D: time course of fluorescence intensity in each cell. Plots of fluorescence intensities (a–d) were obtained from points indicated by arrows in cells a–d in B. E: comparison of amplitudes of the first, second, third, and fourth Ca2+ waves after cessation of stimulation. Ca2+ signal intensities of the second and later waves were normalized to those of the first waves in individual cells. Values are means ± SD of 30 cells. F: comparison of velocities of the first, second, third, and fourth waves. Values are means ± SD of 30 cells. There was no significant difference detected using ANOVA for multigroup comparisons.

 
In Fig. 2B, all Ca2+ waves appeared to stop at the cell boundary. We were interested in whether this is a general feature of waves in guinea pig ventricular muscle. Similar determinations were carried out with 15 preparations. With 178 cells, 497 out of a total of 502 Ca2+ waves observed definitely stopped at the cell boundary. Only 1% of total waves observed appeared to propagate across the boundary, although some of them might, by chance, have appeared to do so. Therefore, it can be concluded that Ca2+ waves did not propagate from cell to cell in intact guinea pig papillary muscle cells.

It has been reported that amplitude, velocity, and frequency of Ca2+ waves depend on Ca2+ content in SR and/or intracellular Ca2+ concentration (1, 13, 21). Because total Ca2+ content of muscle cells was gradually decreasing during rest in guinea pig ventricle (30), it is interesting to know how these parameters of Ca2+ waves change during the resting period. Therefore, we determined peak amplitudes and velocities of Ca2+ waves after cessation of stimulation. The time courses of fluorescence intensities of fluo 3 were determined at points indicated with arrows in individual cells in Fig. 2B and are shown in Fig. 2D. The peak amplitudes of Ca2+ waves looked very similar. Similar determinations were carried out, and the average peak fluorescence intensities of Ca2+ waves are shown in Fig. 2E: they decreased by a small difference with time although the difference was not significant by ANOVA for multigroup comparisons. We can conclude that these decreases were minor, taking bleaching of the dye during the measurements (10% decrease during 33 s) into consideration. Almost the same amplitudes of Ca2+ waves up to the fourth were also obtained with fluo 5F, which had a lower affinity for Ca2+ (KD = 2.3 µM; data not shown).

Wave velocities examined in individual cells were obtained by expanding the time axis of the time-based scan image and determining the slopes of ridges of Ca2+ signal bands (Fig. 2C). With cells where four to five waves occurred during 30 s, the average velocities of the first, second, third, and fourth waves were 131 ± 29, 122 ± 26, 119 ± 33, and 126 ± 38 µm/s (30 cells from 5 preparations), respectively. The first waves propagated significantly faster than the second and third ones when tested by paired t-test (P < 0.05); the difference of ~7–9%, however, was not very large. In fact, it was not significant by ANOVA for multigroup comparison. Therefore, we may conclude that the peak amplitude or velocity of Ca2+ waves does not dramatically change during the period of determination.

The time-based scan image shown in Fig. 2B indicates that the wave interval gradually increased with time. For extensive analysis of wave intervals in each cell, time-based scan images were acquired along the line oriented with a shorter axis of cells as shown by a white line in Fig. 3A, left, thus allowing examination of a large number of cells at once (Fig. 3A, right, also see Figs. 4 and 5). Figure 3B explains how the wave number was defined and how the wave intervals between two consecutive waves were determined. The relationships between wave number and wave interval in individual cells is plotted in Fig. 3C. The wave intervals increased more as waves occurred later in each cell. On closer examination, the wave interval between two adjacent Ca2+ waves, one after the other, increased with a constant ratio as described by the following geometric progression equation:

(1)
where an is an interval between (n – 1)th and nth Ca2+ waves, a1 is an interval between the last Ca2+ transient and the initial Ca2+ wave, r (must be >1) is the increasing ratio of wave interval, and n is the ordinal number of the Ca2+ wave. The fit of data in each cell to Eq. 1 was strikingly good, with an average value of the least square of the fit, R2, of 0.971 ± 0.037 (n = 32) in cells that showed more than three waves in normal Krebs solution. In cells that showed more than two waves within 30 s, values for a1 and r were 0.75–7 s and 1.3–4.2, respectively. Some cells showed no wave or only one after stimulation, where a1 or r is infinitely large. Therefore the first interval (a1) and the increasing ratio (r) values were variable from cell to cell, even in the same preparation. Similar results were obtained with 15 other preparations.



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Fig. 3. Wave intervals after cessation of stimulation. A: time-based scan image obtained from a line transverse to cells in guinea pig papillary muscle (vertical white solid line on left). B: scheme for determination of wave intervals. Intervals were determined as depicted. C: wave interval vs. wave-number relationship. Curves are least-squares fit to Eq. 1. Different symbols indicate different cells.

 


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Fig. 4. Effect of cyclopiazonic acid (CPA), a SERCA inhibitor, on occurrence of Ca2+ waves. A: time-based scan images after cessation of high-frequency stimulation in the presence (b) and absence (a and c) of 10 µM CPA. Image c was obtained after washout of CPA. The muscle fiber was loaded with rhod 2 and observed with a x20 objective. B: wave intervals in the same cell (indicated by arrows in A) before, during, and after treatment of CPA.

 


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Fig. 5. Effect of lowering Na+/Ca2+ exchange activity on Ca2+ wave intervals. A: time-based scan images in the presence of 26 mM Na+ solution. The muscle had been stimulated at 0.5 Hz for 4 min, and then the stimulation was stopped. B: wave intervals in individual cells in the presence of 26 mM Na+. Different symbols indicate different cells. C: time-based scan image obtained in 0Na+/0Ca2+ solution. Before the acquisition of Ca2+ images, the muscle had been stimulated at 2 Hz for 5 min in normal Krebs solution and then incubated in 0Na+/0Ca2+ solution under a resting condition for 10 min. D: wave intervals in individual cells. For this experiment, waves were numbered in order of occurrence during acquisition (from left to right in C). Muscles were loaded with fluo 3. Different symbols indicate different cells.

 
The above results showed that wave amplitudes or velocities changed only slightly. Wave intervals, in contrast, increased one after the other. These findings suggest that, in each cell, the amount of Ca2+ released from SR at wave occurrence is almost constant and that a wave can be triggered when the SR loading reaches a threshold level. Because Ca2+ waves appeared when the background Ca2+ concentration was decreasing (Figs. 1 and 2), the ambient cytoplasmic Ca2+ concentration cannot have been a critical factor. To confirm this idea, we perturbed Ca2+ homeostasis in cardiac cells by modulating Ca2+ uptake by SR and Ca2+ extrusion by Na+/Ca2+ exchanger and examined how wave intervals were affected by them.

In Fig. 4A, Ca2+ wave images were obtained after high-frequency stimulation in the absence and presence of CPA, a SERCA inhibitor. Because intensive inhibition of SERCA activity by CPA resulted in no occurrence of Ca2+ waves (data not shown), the determination in Fig. 4Ab was carried out 10 min after addition of 10 µM CPA, where SERCA inhibition was partial. With this preparation, in the absence of CPA, eight cells showed two or more Ca2+ waves, whereas in the presence of CPA two out of the eight cells showed no wave, one showed only one, and the other cells showed two waves. In the presence of CPA, Eq. 1 was still effective, but both a1 and r were increased (Fig. 4B). With the five cells that showed two waves in the presence of CPA, the averages of a1 increased by 70%, from 4.3 ± 1.4 to 7.4 ± 3.3 s by addition of CPA, whereas r increased by ~50%, from 2.1 ± 0.5 to 3.1 ± 0.6. This means that average intervals increased 2.5-fold (1.7 x 1.5 = 2.55). After washout of CPA, the two parameters were partially reversed (Fig. 4, Ac and B). Wave velocity, on the other hand, was slightly but significantly decreased, from 135 ± 15 (n = 26) to 124 ± 26 (n = 13) µm/s, by addition of CPA. Similar results were obtained with three other preparations. Effect of CPA on Ca2+ wave amplitudes was not examined because fluorescence intensity of fluo 3 itself decreased significantly with time during several series of determinations. The results obtained here suggest that the Ca2+ uptake rate in SR greatly affects Ca2+ wave intervals in contrast to its minor impact on the velocity of the Ca2+ wave.

Next, we examined the effect of reduced Ca2+ extrusion by the Na+/Ca2+ exchange reaction on Ca2+ waves. In the experiment shown in Fig. 5A, the Na+ concentration of Krebs solution was decreased to 26 mM, and muscle was stimulated at 0.5 Hz. At this stimulation frequency, a number of Ca2+ waves were induced in the 26 mM Na+ solution (Fig. 5A), whereas no waves occurred in the normal Krebs solution (cf. Fig. 1). The intervals of Ca2+ waves at 26 mM Na+ are plotted in Fig. 5B. Among 14 cells that showed more than three waves, six cells showed a gradual increase in wave intervals, whereas the rest of the cells did not show clear prolongation. The fit of all results to Eq. 1 was poor, with the average value of the R2 of 0.698 ± 0.206 (n = 14). These results suggest that Ca2+ extrusion via the Na+/Ca2+ exchange reaction contributes to the prolongation of Ca2+ wave intervals. To confirm this idea and to exclude a possible effect of Ca2+ influx from extracellular space, Ca2+ waves were monitored in Na+-free/Ca2+-free solution. In this solution, both Ca2+ influx from the outside and Ca2+ extrusion by the Na+/Ca2+ exchange reaction are inhibited; therefore, Ca2+ content of the muscle is maintained for a long time (3). In the experiment shown in Fig. 5, C and D, the muscle was stimulated at a specified frequency in standard Krebs solution to load SR with Ca2+ to a fixed level and then incubated in 0Na+/0Ca2+ solution at rest for 10 min or more. This means that Ca2+ movement is restricted within the cell. A great number of Ca2+ waves with regular intervals ranging between 0.7 and 5 s were observed in the absence of extracellular Ca2+ or Na+. Under this condition, Ca2+ waves showed no prolongation in their intervals. This means that Ca2+ released from SR during the Ca2+ wave is again taken up to reload SR up to the threshold level. This indicates that the Na+/Ca2+ exchange reaction is most critical for prolongation of Ca2+ waves in physiological solution among the following three candidates: Ca2+ influx, Ca2+ extrusion by the Na+/Ca2+ exchange reaction, and SR Ca2+ pump activity. Therefore the r value may reflect Na+/Ca2+ exchange activity in individual cells. The a1 value, on the other hand, may reflect both Na+/Ca2+ exchange activity and Ca2+ loading level of SR at the last electrical stimulation.

Previous studies with rat ventricular muscles indicated that Ca2+ waves in a mechanically damaged muscle region often showed cell-to-cell propagation (1). In guinea pig ventricle, spontaneous Ca2+ waves were also observed in damaged regions. These damaged cells resulted in one of these alternatives: recovery with eventual disappearance of Ca2+ waves after gradual prolongation or final cell death with sustained elevated cytoplasmic Ca2+ after more frequent Ca2+ waves in several subsequent tens of minutes. Cells showing spontaneous Ca2+ waves are thus considered to be in a state between alive and dead. Interestingly, in the partially damaged muscles, wave propagation often appeared to be unidirectional, with waves traveling from a damaged focus to an intact region (Fig. 6). We examined whether these waves propagate in adjacent cells. Figure 6Aa–Ac shows the case in "Ca2+ paradox" (see MATERIALS AND METHODS), where most Ca2+ waves (marked by white arrowheads in c) traveled from left to right along longitudinal cellular axes, with only a few exceptions (marked by white arrows) in the reverse direction (please refer to the Supplemental Material for this article to view renderings). To know whether those waves actually propagate across the cell boundary under these conditions, time-based scan images from myocytes marked 1–3 in Fig. 6Ad were obtained (Fig. 6Ae–Ag). These images showed that the majority of Ca2+ waves propagated rightward and stopped at the cell boundary (Fig. 6, Ae and Af). Only waves marked with asterisks seemed to propagate across the cell boundary. However, this may be fortuitous because wave frequencies in individual cells were almost regular, although with distinct rates. The Ca2+ wave marked by the arrow in Fig. 6Ag was probably an exceptional case of propagation: it appears that the Ca2+ wave from the right propagated across the cell boundary and stopped when it collided with another wave from the left. Figure 6B shows another example of Ca2+ waves at a region partially damaged by "overload and anoxia" (see MATERIALS AND METHODS). In this muscle, the damage was more heterogeneous (see white cells and their surrounding cells) than that seen in Fig. 6A. In this case, unidirectional Ca2+ waves were also seen in four longitudinally adjacent cells. A time-based scan image revealed that Ca2+ waves propagated leftward, in the direction of decreasing frequency of Ca2+ waves. Similar results were obtained with mechanically damaged muscle (Fig. 6C). We analyzed the direction of propagation of these spontaneous waves to confirm the relationship between the direction and wave frequency. Among 44 cases in 6 preparations that showed spontaneous waves in two longitudinally neighboring cells, 31 cases showed wave propagation in the direction of less frequency, 4 cases in the reverse direction, and 9 cases in either direction. These results indicate that there is a clear tendency in the direction of wave propagation in cells in damaged regions.



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Fig. 6. Spontaneous Ca2+ waves in partially damaged muscles under resting condition; 500 consecutive images were taken every 67 ms without electrical stimulations. A: Ca2+ waves in a muscle subjected to treatment of Ca2+ paradox. a–c: Determination of direction of wave propagation; b was taken at 266 ms after a; c, Ca2+ waves during the first 133 ms are shown in green, and those during the next 133 ms are in red. Note that, in most cells, waves proceeded from left to right (arrowheads), and in only a few cells from right to left (arrows). *Wave initiated within the second 133 ms; d, contour of some cells in a–c; e–g, time-based scan images obtained from lines drawn on adjoining cells (e, f, and g correspond to lines 1, 2, and 3 in d, respectively). Vertical bars above e–g mark the cell boundary. Asterisks in e indicate waves that appeared to propagate across the cell boundary. Arrow in g indicates a wave that appeared to propagate beyond the cell boundary. Note also that this wave showed an exceptional 2-way propagation; h: time-based scan image in Ca2+-free Krebs solution that was taken 5 min after e. B and C: Ca2+ waves in cells damaged by anoxia (B) and cutting (C). a, Images of the damaged muscles. Contoured cells showed spontaneous Ca2+ waves. Bright (white) regions are dead cells that showed neither waves nor electrical activity; b, time-based scan image obtained from a line on adjoining cells. *Waves that appeared to propagate over the cell boundary. Vertical lines indicate the cell boundary. Please refer to the Supplemtal Material for this article (published online at the American Journal of Physiology-Cell Physiology web site) to view a video of Aa–Ac.

 
To examine whether Ca2+ waves in one cell actually do not propagate into the next cell in these regions, we determined the latency period between two successive waves in longitudinally adjacent cells where unidirectional waves were observed. As shown in Fig. 7A, a period between the arrival of one wave at the cell boundary in the cell and the occurrence of a new wave in the next cell was determined. Figure 7B shows a histogram for the latency period. Because of the similar frequency of events at each bin of the period throughout 0~0.8 s, it is concluded that wave occurrences in one cell were not perturbed by Ca2+ waves from neighboring cells. Therefore, these results suggest that very few Ca2+ waves show cell-to-cell propagation, even in regions of unidirectional wave flow.



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Fig. 7. Histogram for latency periods between 2 successive waves at the cell boundary where waves were unidirectionally propagated. A: scheme for determination of the latency. Latency period is the time between the arrival of one wave and the departure of a new wave, as depicted in A. B: histogram with bins of 133-ms increments. Bars represent the no. of waves with each latency. (Data from 168 sets of waves, 13 pairs of cells of which those downstream showed an average wave interval of 0.9 s or longer.) Note that their frequencies were almost the same, irrespective of latency periods.

 
To know what the cause is for the unidirectional propagation of waves, we carried out some preliminary experiments. It has been reported that, at damaged regions, cytoplasmic pH is more acidic with accumulation of lactic acid (8, 10, 11, 25); also, H2O2 or free radical has been shown to be produced during ischemia and reperfusion (27, 34). It is therefore possible that a gradient in one of these factors might underlie the one-way waves. However, an addition of butyrate (20 mM), which has been reported to make intracellular pH acidic (8), slightly lowered wave frequency in 0Na+/0Ca2+ solution, although it prolonged the decay time of Ca2+ waves significantly. Similar results were obtained in an acidic bicarbonate-buffered 0Na+/0Ca2+ solution (pH of 6.7), which was made by bubbling with 30% CO2-70% O2 (data not shown). Lactate (20 mM) or H2O2 (300 µM) also did not increase wave frequency. These results suggest that those factors cannot be the cause of the unidirectional Ca2+ waves. Next, the effects of Ca2+ influx from the outside and extrusion by the Na+/Ca2+ exchanger were examined. When muscles showing spontaneous Ca2+ waves were incubated in Ca2+-free solution, frequency of the waves gradually decreased, although it took more time than intact muscle did. At this point, the direction of waves became two way (see Fig. 6Ah). The one-way Ca2+ waves were also converted to two way if the muscles were incubated in 0Na+/0Ca2+ solution for >10 min, although Ca2+ waves of high frequencies were often converted to a sustained elevation of cytoplasmic Ca2+ in 0Na+/0Ca2+ solution with highly damaged/overloaded muscles (data not shown). Taken together, these results suggest that the gradient in the increased cytoplasmic Ca2+ may be the underlying mechanism for spontaneous and unidirectional Ca2+ waves in a partly damaged region of muscles.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In this investigation of Ca2+ waves in guinea pig papillary muscle, the type of preparation used has the following advantages over single-cell preparations in the study of cardiac muscle function, and led to the conclusions summarized below. 1) Many pieces of information from a large number of individual cells can be obtained at once, and each of them can be easily integrated into the behavior of a mass of muscle. The data in Figs. 14 show clearly that the Ca2+ signals underlying the aftercontraction, which is considered to be a prelude to triggered arrhythmias, consist of asynchronous Ca2+ waves in individual cells. 2) Cells composing papillary muscles turned out to be miscellaneous in their intrinsic properties related to Ca2+ waves and independent of each other (Figs. 1, 3, and 5). This independence of each cell is also the case under such pathological conditions, as shown in Figs. 6 and 7. 3) Only these multicellular preparations allow us to also investigate the interaction between cells under pathological conditions. One-way propagation of waves with partially damaged cells was observed, for which the gradient of cytoplasmic Ca2+ concentration may be the underlying cause (Fig. 6). 4) Ca2+ waves scarcely propagated across the cell boundary, not only of intact cells but also of partially impaired cells (Figs. 2 and 6).

In view of the well-known marked species specificity in Ca2+ homeostasis in heart muscle, the results of this study are valuable because they were obtained with guinea pig ventricular muscles, whereas the previous results (1, 13, 18, 26) were obtained exclusively with rat and mouse heart. There are significant differences in Ca2+ homeostasis in ventricular muscles between rat/mouse and other animals, including guinea pig (3, 29, 30). Postrest contraction is a noticeable example. After a period of rest after steady-state stimulation, the first contraction of rat muscle is largest, followed by decremental ones (negative staircase), whereas guinea pig ventricular muscle shows a positive staircase, being well explained by the change in Ca2+ stored in SR during rest (3, 17).

This study has revealed some differences in the properties of Ca2+ waves between rat and guinea pig ventricular muscles. First, when the muscle was maintained at rest, no Ca2+ wave occurred in guinea pig papillary muscle. In rat ventricular muscle, spontaneous Ca2+ waves were observed at room temperature (1, 13, 18, 22, 33). Second, Ca2+ wave intervals dramatically changed after high-frequency stimulation in guinea pig ventricle, whereas in rat ventricular muscle, frequency of Ca2+ waves remained nearly constant (13). These results can be explained by a large contribution of Na+/Ca2+ exchange reaction in guinea pig ventricle.

In intact guinea pig ventricular muscle, Ca2+ waves seldom propagated beyond the cell boundary. Although a similar conclusion was reached in previous studies with rat hearts, the reported probability with rat heart (3–13% for normal and >20% for Ca2+ overloaded; see Ref. 13) was much greater than that with guinea pig heart (<1%). The reason for this difference is not clear. Because the most important molecule involved in intercellular communication is the gap junction channel, one possibility would be some difference(s) in its number and/or properties.

The luminal Ca2+ levels, cytoplasmic Ca2+ concentrations, and Ca2+ sensitivity of the Ca2+-releasing channels were proposed to be important in the occurrence of Ca2+ waves (1, 13, 21). In this study, we showed that Ca2+ waves were induced after a series of high-frequency stimulations under the condition where the cytoplasmic Ca2+ concentration was gradually decreasing with time (Fig. 1). Wave amplitudes and velocities were not changed much, although the interval between Ca2+ waves was gradually prolonged (Figs. 2 and 3). These results suggest that the loading level of SR was similar when a spontaneous Ca2+ wave occurred, indicating that the luminal Ca2+ level is critically important for induction of Ca2+ waves, as proposed previously (12). The effects of inhibition of SERCA (Fig. 4) and the reduction in the Na+/Ca2+ exchange rate (Fig. 5) on wave intervals further support this conclusion. Ca2+ that has entered via the L-type Ca2+ channel upon electrical stimulation is partly accumulated in the SR by Ca2+-ATPase, is partly extruded out of cells by the Na+/Ca2+ exchanger, and partly stays in cytoplasm during the subsequent resting period. When the Ca2+ loading level in SR reaches the threshold in a cell, spontaneous Ca2+ release occurs. Because SR throughout the entire cell should be loaded with Ca2+ to a similar extent when the wave occurs, Ca2+ release would be easily propagated as waves. Released Ca2+, in turn, is partly taken up to SR by SERCA activity and partly extruded by Na+/Ca2+ exchange reaction from the cell. This extrusion of Ca2+ by the Na+/Ca2+ exchange reaction results in reduction in the cytoplasmic Ca2+, which leads to reduction of the Ca2+ uptake rate in SR and in turn to prolongation of wave intervals. Amplitude and velocity of the next Ca2+ wave, however, remain unaffected.

Time-based scan analysis of consecutive two-dimensional Ca2+ images of the multicellular preparation showed that Ca2+ wave intervals were variable among cells, even in the same preparation (Fig. 3, A–C). This variation was also seen in the absence of Na+/Ca2+ exchange activity (Fig. 5). These results suggest that variations among cells in the ability of Ca2+ handling must be intrinsic properties of the plasma membrane, SR, and/or other systems that directly affect Ca2+ movements or indirectly do so by changing critical modulatory factors, such as ATP concentration, pH, and so on. Cells in multicellular preparations appear to be rather miscellaneous in their characteristics of Ca2+ homeostasis and independent of neighboring cells.

It is interesting that Ca2+ waves often show one-way propagation in a neighboring area in a partly damaged region. This result indicates that Ca2+ waves apparently travel along gradients of some components that tissue degradation has caused. It should be noted, however, that Ca2+ waves seldom propagated across the cellular boundary. Because cells are independent in terms of wave frequency and because cell-to-cell propagation of Ca2+ waves was rarely observed, destruction of cell border or gap junction cannot be the reason. Our preliminary results excluded the possibility of changes in pH or H2O2 level but suggested the gradient of the cytoplasmic Ca2+ concentration as a candidate. It is quite probable that some Ca2+ flow into a neighboring cell during Ca2+ waves through gap junction channels. This Ca2+ influx may contribute to accelerating Ca2+ loading of SR, but it would not induce the Ca2+ wave if the luminal Ca2+ level did not reach the critical level. A possibility of the opening of the L-type Ca2+ channel is less likely because nifedipine was not effective in protecting against Ca2+ paradox (9) or spontaneous Ca2+ waves in our experiments (data not shown), although a possibility of other Ca2+ influx pathways (7, 16) cannot be excluded. Inhibition of the Na+/Ca2+ exchanger, which extrudes Ca2+, is another likely explanation for the increase in cytoplasmic Ca2+. A factor of resting membrane potential must also be considered as a reason for the reduced Na+/Ca2+ exchange rate. A more depolarized membrane potential, which is expected in damaged cells, would be unfavorable to Ca2+ extrusion by the Na+/Ca2+ exchanger. The electrotonic spatial distribution of depolarized membrane potential at the damaged region would help form the gradient of cytoplasmic Ca2+ concentration.

The decrease in or loss of the conductance of gap junction channels might be a cause for infrequently occurring cell-to-cell propagation of Ca2+ waves in temporarily Ca2+-overloaded intact muscles or partially damaged muscles, because their permeability is reported to be decreased by high intracellular Ca2+ or low pH (3, 32). In intact muscles, the possibility of inactivation of the gap junction is excluded because whole muscle bundles ~3 mm long were excited by a localized stimulation with a small electrode of ~50 x 20 µm in size (data not shown). Based on this and previous observations (32), it seems reasonable to assume that functional gap junctions were well maintained in the experiments shown in Figs. 15. In partially damaged muscles, it was difficult to estimate the function of gap junctions from electrical conductivity because the cells themselves had already become less responsive to field electrical stimulation. The conductance of gap junctions might be somewhat lowered, since the previous report showed that an exposure of calf ventricular muscle to a highly toxic dose of ouabain gradually decreased the conduction to ~60% of the initial after 90 min (32). However, the reduced conductance of the gap junction, if any, may not be the main reason for the scarce cell-to-cell propagation, because occurrence of the unidirectional wave is well explained by ionic or electronic conductivity of gap junctions, as described above. In either event, it would be interesting to investigate how the increased basal Ca2+ or long-lasting occurrence of Ca2+ waves modulates gap junctions.

In conclusion, we concentrated on whether Ca2+ waves propagate beyond a cell boundary and found that their cell-to-cell propagation was infrequent not only in intact cardiac muscles but also in partially damaged ones. During the course of the experiments, we also found unidirectional Ca2+ waves in partially damaged muscles. Because this phenomenon may be involved in the prognosis for a partially injured heart, the relation between Ca2+ waves and spread of damage in a multicellular preparation requires further investigation.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by Grants-in-Aid to Y. Ogawa (no. 11470026) and N. Kurebayashi (no. 13670096) and a High Technology Research Center Grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    ACKNOWLEDGMENTS
 
We are grateful to Professor Emeritus Hiroshi Yamaguchi, Department of Cardiology, Juntendo University School of Medicine, for arranging our collaboration on this study. We thank Mei Ling and Yuriko Yotsui for helpful assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Kurebayashi, Dept. of Pharmacology, Juntendo Univ. School of Medicine, 2–1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan (E-mail: nagomik{at}med.juntendo.ac.jp)

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.

1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00200.2004/DC1/. Back


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