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(Circulation Research. 2000;86:1093.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Three Distinct Types of Ca²⁺ Waves in Langendorff-Perfused Rat Heart Revealed by Real-Time Confocal Microscopy

Tomoyuki Kaneko¹, Hideo Tanaka¹, Masahito Oyamada, Satoshi Kawata, Tetsuro Takamatsu

From the Departments of Pathology and Cell Regulation (T.K., M.O., T.T.) and Laboratory Medicine (H.T.), Kyoto Prefectural University of Medicine, Kyoto, and the Department of Applied Physics (T.K., S.K.), Osaka University Graduate School of Engineering, Suita, Japan.

Correspondence to Tetsuro Takamatsu, Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-Ku, Kyoto 602-0841, Japan. E-mail ttakam{at}basic.kpu-m.ac.jp

	Abstract

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Abstract—Although Ca²⁺ waves in cardiac myocytes are regarded as arrhythmogenic substrates, their properties in the heart in situ are poorly understood. On the hypothesis that Ca²⁺ waves in the heart behave diversely and some of them influence the cardiac function, we analyzed their incidence, propagation velocity, and intercellular propagation at the subepicardial myocardium of fluo 3–loaded rat whole hearts using real-time laser scanning confocal microscopy. We classified Ca²⁺ waves into 3 types. In intact regions showing homogeneous Ca²⁺ transients under sinus rhythm (2 mmol/L [Ca²⁺]_o), Ca²⁺ waves did not occur. Under quiescence, the waves occurred sporadically (3.8 waves · min^-1 · cell^-1), with a velocity of 84 µm/s, a decline half-time (t_1/2) of 0.16 seconds, and rare intercellular propagation (propagation ratio <0.06) (sporadic wave). In contrast, in presumably Ca²⁺-overloaded regions showing higher fluorescent intensity (113% versus the intact regions), Ca²⁺ waves occurred at 28 waves · min^-1 · cell^-1 under quiescence with a higher velocity (116 µm/s), longer decline time (t_1/2=0.41 second), and occasional intercellular propagation (propagation ratio=0.23) (Ca²⁺-overloaded wave). In regions with much higher fluorescent intensity (124% versus the intact region), Ca²⁺ waves occurred with a high incidence (133 waves · min^-1 · cell^-1) and little intercellular propagation (agonal wave). We conclude that the spatiotemporal properties of Ca²⁺ waves in the heart are diverse and modulated by the Ca²⁺-loading state. The sporadic waves would not affect cardiac function, but prevalent Ca²⁺-overloaded and agonal waves may induce contractile failure and arrhythmias.

Key Words: Ca²⁺ wave • confocal microscopy • intercellular propagation • Langendorff-perfused heart

	Introduction

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The elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac contractile activities originates from elementary sarcoplasmic reticulum (SR) Ca²⁺-release events, called Ca²⁺ sparks.^{1 2 3} During membrane depolarization in the action potentials, simultaneous summation of Ca²⁺ sparks produces spatially uniform elevation of [Ca²⁺]_i throughout the cells, ie, Ca²⁺ transients. Conversely, under unstimulated conditions, recruitment of Ca²⁺ sparks produces spontaneous localized elevation of [Ca²⁺]_i, which propagates within myocytes, especially under Ca²⁺ overload. This propagating [Ca²⁺]_i elevation, originally observed as a wavelike motion of contractile bands in cardiac myocytes,^{4 5 6} has been called the Ca²⁺ wave^{7 8} and is regarded as the origin of oscillatory membrane potentials leading to triggered arrhythmias.⁹ For the past decade, the properties of Ca²⁺ waves in cardiac myocytes have been studied precisely after the advent of fluorescent Ca²⁺ indicators^{7 8 9 10 11 12 13} and laser scanning confocal microscopy.^{11 12 13}

Despite ample information on Ca²⁺ waves,^{7 8 9 10 11 12 13} the role of the waves in the heart in situ is poorly understood. This is because Ca²⁺ waves have been studied mostly in enzymatically isolated cells. Minamikawa et al¹⁴ first demonstrated that Ca²⁺ waves occur in the perfused rat whole heart. However, their detailed quantitative properties were not assessed because of the low temporal resolution of the X-Y images. We have developed a system for in situ imaging of [Ca²⁺]_i equipped with a multipinhole-type confocal scanning device, which enabled us to visualize real-time X-Y images of Ca²⁺ waves.^{15 16} Using this system on Langendorff-perfused rat hearts with simultaneous recording of electrocardiograms, we found that Ca²⁺ waves were completely abolished by ventricular excitation, suggesting that the waves in the whole heart play little, if any, pathophysiological role.¹⁵ Nevertheless, it is possible that Ca²⁺ waves have some aggravating role on cardiac function if they occur frequently and propagate beyond individual cells on a large scale under certain Ca²⁺-overloaded conditions. In this regard, quantitative analysis of Ca²⁺ waves in the working whole heart is essential to understand their functional significance.

We postulated that Ca²⁺ waves in the whole heart exhibit various distinct frequencies, propagation velocities, and intercellular propagations, all of which may depend on the degree of Ca²⁺ overload and electrical activities of the heart, ie, Ca²⁺ waves may occur more frequently and propagate more prevalently to the surrounding cells under highly Ca²⁺-overloaded conditions. To examine these hypotheses, we used real-time confocal microscopy to conduct quantitative analyses of Ca²⁺ waves on the perfused rat whole hearts, focusing on spatiotemporal occurrence and intercellular propagation under both the intact and Ca²⁺-overloaded conditions.

	Materials and Methods

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The preparation of the fluo 3-AM–loaded rat hearts and the confocal system were identical to those described previously.¹⁵ The experiments were conducted on 16 male Wistar rats (9 weeks old) at 23°C to 25°C under Langendorff perfusion (5 mL/min) with Tyrode’s solution ([Ca²⁺]_o=2 mmol/L, unless otherwise specified). The confocal plane was optimized with a glass coverslip (170-µm thickness) placed gently on the heart. The motion artifact on the image was prevented by 20 mmol/L 2,3-butanedione monoxime (BDM), which was added to the perfusate. The ECG was recorded through silver wires (0.3-mm diameter) inserted into the apical region of the heart. Unless otherwise specified, Ca²⁺ waves were analyzed under quiescence via blockade of atrioventricular conduction produced by mechanical ablation of the atrioventricular junction. Some hearts were electrically stimulated through the silver wires. In some experiments, a glass microelectrode (tip diameter <10 µm) was inserted into localized regions to apply damage to the myocardium.

Analyses of images were conducted on digitized fluorescent signals (512x480 pixels) stored on a hard disk every 33 ms. Line-scan images were reconstructed by cumulatively layering a series of consecutive X-Y image frames scanned according to the line along the path of the waves. The propagation velocity was calculated from the slope of the line-scan image. The profile of Ca²⁺ waves was obtained by averaging the line-plot data of line-scan images from 5 to 7 adjoining scan lines at the middle of the cells along the long axis. The fluorescence intensity (FI) in a region of interest (ROI) (FI_ROI) was obtained by averaging the FI value of 250 pixels during diastole. With regions with homogeneous Ca²⁺ transients but no frequent Ca²⁺ waves regarded as intact, FI was estimated by use of a value obtained from FI_ROI relative to that in the neighboring intact region (FI_intact), ie, FI_ROI/FI_intact. The incidence of Ca²⁺ waves and their intercellular propagation ratio (R_prop) were analyzed from continuously recorded video images. R_prop was defined as the ratio of the number of waves propagating to the adjacent cells over the total number of waves in an ROI for 5 minutes. The quantitative data (mean±SD) were statistically analyzed by ANOVA, and significance at P<0.05 was defined by Fisher’s test.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Ca²⁺ Waves in the Intact Region
Figure 1A shows fluo 3-fluorescent images on a subepicardial region of the perfused heart at 2 mmol/L [Ca²⁺]_o. The Ca²⁺ transients occurred simultaneously with QRS waves, whereas no significant fluorescent elevation occurred during diastole. Similar findings were obtained in 12 other hearts with regular rhythm at 64±12 bpm. When the ventricular excitation rate was decreased (<5 bpm) by blockade of atrioventricular conduction (n=9), Ca²⁺ waves appeared (Figure 1B, 1 through 5) sporadically at a mean frequency of 3.8±2.4 (0.8 to 14.0) waves · min^-1 · cell^-1 (70 cells in 9 hearts) and were abolished by a spontaneous Ca²⁺ transient (6 through 10). Ca²⁺ transients evoked by electrical stimulation also abolished Ca²⁺ waves (not shown).

View larger version (74K): [in this window] [in a new window]   Figure 1. Real-time confocal fluo 3-fluorescent images of the subepicardial myocardium in the perfused heart at 2 mmol/L [Ca2+]o. Images are shown by pseudocolors corresponding to the FI. A, Sequential X-Y images (displayed every 330 ms) under simultaneous ECG. Ca2+ transients (1, 5, and 9) occurred simultaneously with QRS waves. B, Sporadic appearance (1 to 5) and Ca2+ transient–induced cancellation (6 to 10) of Ca2+ waves during quiescence produced by the atrioventricular conduction blockade. The Ca2+ transient in frame 6 occurred spontaneously. C, Sequential X-Y images of Ca2+ waves (every 100 ms): unidirectional (a) and bidirectional (b) propagation, and collision (c). D, Corresponding line-scan images of the waves in C along the arrows in frame 1. E, Time course of the relative FI for Ca2+ wave with the difference of FI between baseline and peak normalized.

Ca²⁺ waves in the perfused hearts exhibited intracellular propagation similar to those in isolated myocytes studied previously.^{7 8 9 10 11 12 13} Sequential X-Y images at 2 mmol/L [Ca²⁺]_o showed that the waves propagated along the longitudinal axis of the cells (Figure 1C). The corresponding line-scan images (Figure 1D) revealed that the waves proceeded at a constant velocity of 90 µm/s in 1 (a) or 2 (b) directions. In some cases, 2 waves collided within single cells and were subsequently annihilated (c). Ca²⁺ waves propagated longitudinally at 84±16 (52 to 129) µm/s, either unidirectionally or bidirectionally to the cell boundaries (n=73). The waves showed a monotonic decline in their fluorescent profile (Figure 1E) similar to that in isolated myocytes.¹⁷ After a quick rise, the waves declined with a decay half-time (t_1/2) of 0.16±0.10 seconds (n=60). As for the intercellular propagation of Ca²⁺ waves, the waves in the perfused heart barely transmitted to the surrounding myocytes. Of 320 waves, 12 showed propagation to the adjacent myocyte with an R_prop of 0.034. Hereafter, we call these waves in the intact regions sporadic waves.

[Ca²⁺]_o and Stimulus Dependence of the Sporadic Ca²⁺ Waves
The incidence of sporadic waves increased at higher [Ca²⁺]_o (Figure 2A). At 4 or 6 mmol/L, 2 Ca²⁺ waves often collided and subsequently annihilated within single cells. The waves at higher [Ca²⁺]_o showed a tendency to propagate with higher velocity (Figure 2B) and were also abolished by a Ca²⁺ transient. At such higher [Ca²⁺]_o, the waves occurred even between repetitive excitations at 1 Hz (not shown). Even at 6 mmol/L [Ca²⁺]_o, Ca²⁺ waves barely showed intercellular propagation (R_prop=0.057, n=35).

View larger version (46K): [in this window] [in a new window]   Figure 2. [Ca2+]o-dependent incidence (A, *P<0.05) and velocity (B, *P<0.05 vs value at 1 mmol/L [Ca2+]o) of the sporadic waves from 4 hearts. The numbers of waves examined are shown in parentheses. C, Time course of sporadic wave incidence after electrical stimulation. Numbers of Ca2+ waves in an ROI (12 cells) are plotted against time (every 10 seconds) after cessation of 30-second electrical stimuli at 2 mmol/L (left) and 4 mmol/L (right) [Ca2+]o. x, , and • indicate numbers of waves for 10 seconds at 1, 2, and 3 Hz, respectively.

The sporadic waves occurred according to previous excitation. In one representative region (Figure 2C, data composed of 12 cells), the waves appeared after excitation by 30-second consecutive electrical stimulation. At 2 mmol/L [Ca²⁺]_o, they appeared within 30 seconds after 1-Hz stimuli (x) and occurred more frequently over time until the steady state 70 seconds after stimuli (left). As the stimulation frequency increased ( for 2 Hz, • for 3 Hz), the latency for the first appearance of the waves shortened and the waves occurred more frequently. The latency period also shortened when [Ca²⁺]_o increased. At 4 mmol/L [Ca²⁺]_o, stimuli at 2 Hz produced a tremendous number of Ca²⁺ waves immediately after stimuli and a subsequent gradual decrease in incidence (right). Such robust Ca²⁺ waves barely propagated to the adjacent myocytes. Similar stimulation dependence of the waves was obtained in 5 other hearts.

Frequent Occurrence of Ca²⁺ Waves in Regions With Higher FI
In contrast to the sporadic waves, high-frequency Ca²⁺ waves were observed in some specific regions with higher FI (hereafter called Ca²⁺-overloaded waves). Figure 3 shows sequential X-Y images (Figure 3A) and the corresponding line-scan images (Figure 3B) of Ca²⁺-overloaded waves: failure of propagation (Figure 3B-a) and propagation with (Figure 3B-b) and without (Figure 3B-c) delay. These particular waves occurred at 10 waves · min^-1 · cell^-1. Adjacent to the cell with the waves, there was a region with higher static FI (upper left). These Ca²⁺-overloaded waves showed a longer fluorescent profile than the sporadic waves (Figure 3C). Of 60 Ca²⁺-overloaded waves examined, the profile declined significantly slowly (t_1/2=0.41±0.20 seconds, P<0.01).

View larger version (97K): [in this window] [in a new window]   Figure 3. A, Intercellular propagation of Ca2+-overloaded waves (1 through 10) displayed every 100 ms with pseudocolors. Left, Location of waves and direction of propagation (arrows). B, Corresponding line-scan images (bottom) show failure of propagation (a) and propagation with (b) and without (c) delay. C, Comparison of fluorescent profiles of sporadic (dotted line) and Ca2+-overloaded (solid line) waves. D, Sequential X-Y images (every 100 ms) of Ca2+ waves in a region in which mechanical damage was applied via a microelectrode. Ca2+ wave initiated at center of frame shows transverse (4 through 7) and longitudinal (5 through 10) propagation to adjacent cells. Schematic representation beneath each image.

Ca²⁺-overloaded waves were also observed in a region in which mechanical damage was caused by a glass microelectrode (n=5). Figure 3D shows the waves in the damage-applied region. They occurred frequently, at 48 waves · min^-1 · cell^-1. One Ca²⁺ wave at the center propagated to the adjacent cells transversely (4 to 7) and longitudinally (5 to 10). Similar patterns of intercellular wave propagation were observed in 31 regions of 7 hearts. The Ca²⁺-overloaded waves occurred in regions with higher basal FI (FI_ROI/FI_intact=1.13±0.05, n=22, P<0.01). They occurred at 28.1±8.3 waves · min^-1 · cell^-1 (n=40, P<0.01) with a velocity of 116.7±29.4 µm/s (n=63, P<0.01), more frequently and more quickly than those for the sporadic waves at 6 mmol/L [Ca²⁺]_o. The R_prop in such Ca²⁺-overloaded regions was higher (0.23, n=467) than that in intact regions (P<0.01).

The third class of Ca²⁺ waves we examined were extremely high-frequency waves showing ripple-like wave fronts, which disappeared within 10 minutes, resulting in regions with high static FI and no response to electrical stimulation, indicating cell death (hereafter, agonal waves). In a typical example shown in Figure 4A, the waves occurred very frequently at 280 waves/min, as calculated from the line-scan image (C), and showed no propagation to the adjacent cells. Of 12 regions having isolated Ca²⁺ waves as in Figure 4A, 11 waves showed no intercellular propagation (R_prop, 0.08). In the clusters composed of 2 waves, the waves barely propagated intercellularly (R_prop, 0.09; n=93). In such specific regions, electrical stimulation failed to induce Ca²⁺ transients, and Ca²⁺ transients around the regions failed to abolish the waves (not shown). Such high-frequency waves were also observed in regions damaged by microelectrodes (n=5). The agonal waves occurred in regions with higher FI (FI_ROI/FI_intact=1.24±0.09, n=18, P<0.01), showing a frequency of 133.1±65.4 waves · min^-1 · cell^-1 (n=37) and a propagation velocity of 112±25.1 µm/s (n=37).

View larger version (46K): [in this window] [in a new window]   Figure 4. Highly frequent Ca2+ waves (agonal waves). A, Sequential X-Y images (every 100 ms) showing multiple wave fronts originating from 1 focus (left). B and C, Corresponding schematic representation and line-scan image of the waves, respectively.

Figure 5 summarizes the 3 types of Ca²⁺ waves described above. The relative FI (FI_ROI/FI_intact) (Figure 5A) and the incidence of the waves (Figure 5B) showed clear differences among these regions. The propagation velocities of the Ca²⁺-overloaded and agonal waves were higher than those of the sporadic waves (Figure 5C). The R_prop was higher for the Ca²⁺-overloaded waves, whereas the sporadic and agonal waves showed poor intercellular propagation (Figure 5D).

View larger version (37K): [in this window] [in a new window]   Figure 5. Summary of the 3 distinct Ca2+ waves. A, Relative FI (FIROI/FIintact). B, Incidence. C, Propagation velocity. D, Propagation ratio (Rprop). Differences in Rprop were assessed by Fisher’s exact probability test. Numbers of waves examined are shown in parentheses. *P<0.05.

	Discussion

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By focusing on incidence, we revealed the existence of 3 distinct types of Ca²⁺ waves in the subepicardial regions of the perfused heart. These 3 types of waves showed different propagation velocities, relative FIs (FI_ROI/FI_intact), and intercellular propagation ratios (R_prop). The incidence and velocity of Ca²⁺ waves are considered to reflect the difference in [Ca²⁺]_i-loading states in the perfused heart, analogously to their [Ca²⁺]_i dependence in isolated myocytes.^{5 7 12 13} The relative FI can also be regarded as a reliable indicator of the [Ca²⁺]_i-loading state, for the following reasons: (1) the transcoronary perfusion of fluo 3-AM attained homogeneous fluo 3 loading at ROIs,^{14 15} (2) a motion artifact on the image was absent, and (3) there was no noticeable difference in size among the cells with different FI. Therefore, the 3 types of waves we identified are likely to originate from different [Ca²⁺]_i-loading states. In other words, as the myocardial tissue becomes Ca²⁺-overloaded depending on the background conditions of myocytes (ie, functioning normally, Ca²⁺-overloaded and functional, and totally nonfunctional), the properties of Ca²⁺ waves change progressively and successively.

We demonstrate here for the first time that Ca²⁺ waves did not occur in apparently intact regions during sinus rhythm, even at supraphysiological [Ca²⁺]_o (2 mmol/L) and room temperatures, according to real-time images of the waves (Figure 1A). Previously, Ca²⁺ waves were identified in intact multicellular ventricular preparations^{18 19} and perfused intact whole hearts in physiological [Ca²⁺]_o of 1 mmol/L.¹⁴ However, it has been unclear whether or not Ca²⁺ waves occur in the working heart under physiological conditions. This is because previous studies were conducted under arrested conditions.^{14 18 19} We further observed that the waves were abolished by Ca²⁺ transients (Figure 1B) and recurred with latency (Figure 2C). Therefore, it seems reasonable to consider that repetitive excitation of the myocardium prevents the hearts from producing sporadic Ca²⁺ waves.

In contrast to sporadic Ca²⁺ waves, Ca²⁺-overloaded waves occurred frequently, even during repetitive excitation at 2 mmol/L [Ca²⁺]_o. A higher basal FI (Figure 5A) and patchy distribution of cells with high static FI (Figures 3A and 3D) indicated that the regions were likely to be Ca²⁺-overloaded. Ca²⁺-overloaded conditions in the regions were also suggested by the findings that the incidence and velocity of the Ca²⁺-overloaded waves at 2 mmol/L [Ca²⁺]_o (Figure 5B and 5C) were much higher than those of the sporadic waves at 6 mmol/L [Ca²⁺]_o (Figure 2A and 2B). The Ca²⁺-overloaded conditions were probably caused by some inevitable localized injury or damage, which occurred spontaneously but rarely during the preparation of Langendorff perfusion. Local damage by microelectrodes induced the same types of Ca²⁺ waves (Figure 3D), indicating that the waves occur under Ca²⁺ overload. The longer decline phase of the waves (Figure 3C) may also indicate Ca²⁺ overload, because previous reports demonstrated that the decline phase of Ca²⁺ transients was prolonged in damaged or failing myocytes^{20 21} and that Ca²⁺ waves and Ca²⁺ transients showed similar fluorescent profiles.¹⁷ Such prolongation of Ca²⁺ wave profiles may be attributed to some impairment of Ca²⁺ handling, especially reuptake of Ca²⁺ by SR, which is known to be impaired via alteration of phospholamban/SR Ca²⁺ (SERCA) pump activity in damaged or failing hearts.^{22 23} Direct evidence for the SERCA pump modulation of Ca²⁺ waves was provided in the phospholamban-deficient mouse heart, which lacks the inhibitory action of the SERCA pump, by demonstrating that Ca²⁺ waves declined faster.²⁴

We found that 20% of myocytes with the Ca²⁺-overloaded waves had the ability to propagate to adjacent myocytes. However, the waves showed no widespread propagation to the surrounding myocytes: at most, to 3 to 4 adjacent ones. These findings are in agreement with the results of Lamont et al,¹⁹ who reported that 13% of waves propagated to the adjacent cells in the rat ventricular trabeculae. Two factors can be considered to be determinants for intercellular propagation: the inducibility of Ca²⁺ waves (ie, ability of regenerative propagation) and conductivity of the gap junctions. We consider that the limiting step for the observed intercellular wave propagation resides in the former rather than the latter. This is because the gap junctional conductance in the Ca²⁺-overloaded regions is lower than that in the intact regions, according to its [Ca²⁺]_i dependence.²⁵ The importance of the inducibility of Ca²⁺ waves in the adjacent (recipient) myocytes is supported by several previous reports. Lipp and Niggli¹³ proposed that the Ca²⁺-loading state of SR determines the propagation distance of the wave by modulating the degree of positive feedback of SR Ca²⁺ release. Wier et al¹⁸ also regarded the positive feedback of SR Ca²⁺ release as an important factor for wave propagation, because they observed occasional truncation of the waves in the middle of the cells of the intact rat ventricular trabeculae. Lamont et al¹⁹ reported that the waves in rat ventricular trabeculae occasionally propagated to the adjacent cells but aborted in the middle of the cells (called Ca²⁺ spritz), suggesting that the adjacent (recipient) cells are important to complete wave propagation. Taken together, Ca²⁺ waves from the adjacent cells would therefore propagate to the recipient cells only when the cells exert higher positive feedback of SR Ca²⁺ release to induce Ca²⁺ waves. The positive feedback of SR Ca²⁺ release does not proceed very far, even in the Ca²⁺-overloaded regions.

The agonal waves occurred in regions with higher FI than the regions with the Ca²⁺-overloaded waves, suggesting highly Ca²⁺-overloaded conditions. Although the frequent and ripple-like fluctuations are often observed in single isolated myocytes just before they go into contracture (ie, an agonal state), we provide, for the first time, detailed information on the agonal waves in the whole heart. The waves showed little propagation to adjacent cells despite their highly frequent occurrence (ie, high positive feedback of SR Ca²⁺ release). This was due to collision of multifocal waves with subsequent refractoriness and possible closure of the gap junction by presumably higher [Ca²⁺]_i.²⁵ A decrease in the gap junctional conductivity in the agonal regions was suggested by observations that electrical stimulation failed to induce Ca²⁺ transients and that the surrounding Ca²⁺ transients failed to abolish the waves. Another interesting finding regarding agonal waves was that the propagation velocities were not higher than but rather almost equal to those of Ca²⁺-overloaded waves (Figure 5C). Although we have no direct evidence to explain this finding, a refractoriness of Ca²⁺ release from SR in the extremely high-frequency waves may attenuate the increase in the propagation velocity by facilitating the use-dependent inactivation of ryanodine receptors²⁶ at extremely high frequency.

We have to recognize several limitations in this study. First, Langendorff perfusion with a colloid-free solution can produce edema and minimal focal myocardial degeneration.²⁷ Second, a relatively higher [Ca²⁺]_o (2 mmol/L) at room temperature could render the heart Ca²⁺-overloaded, even in an apparently "intact" region. Such conditions potentially augment the occurrence of Ca²⁺ waves. The third limitation resulted from the use of BDM at a relatively higher concentration (>10 mmol/L), possibly precluding the occurrence of Ca²⁺ waves in the perfused heart through its direct effects on Ca²⁺ handling.²⁸ Nevertheless, these limitations do not affect our conclusion that 3 distinct types of Ca²⁺ waves occur in the perfused whole heart, depending on the Ca²⁺-loading state.

Although direct evidence for the pathological significance of Ca²⁺ waves is still lacking, the following speculations can be made from our present whole-heart data. The Ca²⁺-overloaded and agonal waves are likely to have pathological roles. Because Ca²⁺ waves produce arrhythmogenic depolarization in single myocytes,⁹ the frequent and prevalent Ca²⁺-overloaded waves with intercellular propagation may provoke abnormal depolarization of cardiac tissue leading to contractile failure or triggered arrhythmia. The agonal regions may become origins of reentrant arrhythmias when they hamper electrical conduction. In turn, the loss of intercellular propagation of the agonal waves may serve as a protective mechanism against the spatial progression of myocardial damage. Although the pathogenesis of Ca²⁺ overload in the observed regions was not determined, the divergent properties of Ca²⁺ waves revealed in this study may represent certain aspects of the waves that occur under certain pathological conditions, such as myocardial ischemia/reperfusion injury and infarction. Delineation of the functional properties of Ca²⁺ waves under such specific pathological states is open for future research.

	Acknowledgments

 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and from the Future Plan of Japan Society for Promotion of Science. We thank Dr Hideo Kusuoka for reviewing the manuscript and Daniel Mrozek for English editing.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received January 17, 2000; accepted March 30, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Cheng H, Lederer WJ, Cannel MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993;262:740–744.[Abstract/Free Full Text]

2. Lipp P, Niggli E. A hierarchical concept of cellular and subcellular Ca²⁺ signalling. Prog Biophys Mol Biol. 1996;65:265–296.[Medline] [Order article via Infotrieve]

3. Wier WG, Balke CW. Ca²⁺ release mechanisms, Ca²⁺ sparks, and local control of excitation-contraction coupling in normal heart muscle. Circ Res. 1999;85:770–776.[Free Full Text]

4. Rieser G, Sabbadini R, Paolini P, Fry M, Inesi G. Sarcomere motion in isolated cardiac cells. Am J Physiol. 1979;236:C70–C77.[Abstract/Free Full Text]

5. Kort AA, Capogrossi MC, Lakatta EG. Frequency, amplitude, and propagation velocity of spontaneous Ca⁺⁺-dependent contractile waves in intact adult rat cardiac muscle and isolated myocytes. Circ Res. 1985;57:844–855.[Abstract/Free Full Text]

6. Capogrossi MC, Houser SR, Bahinski A, Lakatta EG. Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential. Circ Res. 1987;61:498–503.[Abstract/Free Full Text]

7. Takamatsu T, Wier WG. Calcium waves in mammalian heart: quantification of origin, magnitude, and velocity. FASEB J. 1990;4:1519–1525.[Abstract]

8. Ishide N, Urayama T, Inoue K, Komaru T, Takishima T. Propagation and collision characteristics of calcium waves in rat myocytes. Am J Physiol. 1990;259:H940–H950.[Abstract/Free Full Text]

9. Berlin JR, Cannel MB, Lederer WJ. Cellular origins of transient inward current in cardiac myocytes: role of fluctuations and waves of elevated intracellular calcium. Circ Res. 1989;65:115–126.[Abstract/Free Full Text]

10. Wier WG, Cannel MB, Berlin JR, Marban E, Lederer WJ. Cellular and subcellular heterogeneity of intracellular calcium concentration in single heart cells revealed by fura-2. Science. 1987;235:325–328.[Abstract/Free Full Text]

11. Takamatsu T, Minamikawa T, Kawachi H, Fujita S. Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy. Cell Struct Funct. 1991;16:341–346.[Medline] [Order article via Infotrieve]

12. Williams DA, Delbridge LM, Cody SH, Harris PJ, Morgan TO. Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy. Am J Physiol. 1992;262:C731–C742.[Abstract/Free Full Text]

13. Lipp P, Niggli E. Modulation of Ca²⁺ release in cultured neonatal rat cardiac myocytes. Insight from subcellular release patterns revealed by confocal microscopy. Circ Res. 1994;74:979–990.[Abstract/Free Full Text]

14. Minamikawa T, Cody SH, Williams DA. In situ visualization of spontaneous calcium waves within perfused whole heart by confocal imaging. Am J Physiol. 1997;272:H236–H243.[Abstract/Free Full Text]

15. Hama T, Takahashi A, Ichihara A, Takamatsu T. Real time in situ confocal imaging of calcium wave in the perfused whole heart of the rat. Cell Signal. 1998;10:331–337.[Medline] [Order article via Infotrieve]

16. Takamatsu T. Confocal microscopy: applications in research and practice of pathology. Anal Quant Cytol Histol. 1998;20:529–532.[Medline] [Order article via Infotrieve]

17. Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol. 1996;270:C148–C159.[Abstract/Free Full Text]

18. Wier WG, ter Keurs HEDJ, Marbán E, Gao W, Balke CW. Ca²⁺ "sparks" and waves in intact ventricular muscle resolved by confocal imaging. Circ Res. 1997;81:462–469.[Abstract/Free Full Text]

19. Lamont C, Luther PW, Balke CW, Wier WG. Intercellular Ca²⁺ waves in rat heart muscle. J Physiol (Lond). 1998;512:669–676.[Abstract/Free Full Text]

20. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, Morgan, JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res. 1987;61:70–76.[Abstract/Free Full Text]

21. Beuckelmann DJ, Näbauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992;85:1046–1055.[Abstract/Free Full Text]

22. Pagani ED, Alousi AA, Grant AM, Older TM, Dziuban SW, Allen PD. Changes in myofibrillar content and Mg-ATPase activity in ventricular tissues from patients with heart failure caused by coronary artery disease, cardiomyopathy, or mitral valve insufficiency. Circ Res. 1988;63:380–385.[Abstract/Free Full Text]

23. Misquinta CM, Mack DP, Grover AK. Sarco/endoplasmic reticulum Ca²⁺ (SERCA)-pumps: link to heart beats and calcium waves. Cell Calcium. 1999;25:277–290.[Medline] [Order article via Infotrieve]

24. Hüser J, Bers DM, Blatter LA. Subcellular properties of [Ca²⁺]_i transients in phospholamban-deficient mouse ventricular cells. Am J Physiol. 1998;274:H1800–H1811.[Abstract/Free Full Text]

25. Noma A, Tsuboi N. Dependence of junctional conductance of proton, calcium and magnesium ions in cardiac paired cells of guinea pig. J Physiol (Lond). 1987;382:193–211.[Abstract/Free Full Text]

26. Sham JSK, Song L-S, Chen Y, Deng L-H, Stern MD, Lakatta EG, Cheng H. Termination of Ca²⁺ release by a local inactivation of ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci U S A. 1998;95:15096–15101.[Abstract/Free Full Text]

27. Monticello TM, Sargent CA, McGill JR, Barton DS, Grover GJ. Amelioration of ischemia/reperfusion injury in isolated rats hearts by the ATP-sensitive potassium channel opener. Cardiovasc Res. 1996;31:93–101.[Medline] [Order article via Infotrieve]

28. Backx PH, Gao W-D, Azan-Backx MD, Marban E. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca²⁺]_i and cross-bridge kinetics. J Physiol (Lond). 1994;476:487–500.[Abstract/Free Full Text]

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