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(Circulation Research. 1997;81:462-469.)
© 1997 American Heart Association, Inc.

Articles

Ca²⁺ `Sparks' and Waves in Intact Ventricular Muscle Resolved by Confocal Imaging

Withrow Gil Wier, Henk E. D. J. ter Keurs, Eduardo Marban, Wei Dong Gao, , C. William Balke

From the Department of Physiology (W.G.W., C.W.B.) and the Division of Cardiology, Department of Medicine (C.W.B.), University of Maryland School of Medicine, Baltimore; the Departments of Medicine, Physiology, and Biophysics (H.E.D.J. ter K.), The University of Calgary (Canada); and the Division of Cardiology, Department of Medicine (E.M., W.D.G.), The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Dr W.G. Wier or Dr C.W. Balke, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore St, Baltimore, MD 21201. E-mail gwier001@umabnet.ab.umd.edu or bbalke{at}heart.ab.umd.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The [Ca²⁺]_i transient in heart is now thought to involve the recruitment and summation of discrete and independent "units" of Ca²⁺ release (Ca²⁺ "sparks") from the sarcoplasmic reticulum, each of which is controlled locally by single coassociated L-type Ca²⁺ channels ("local control theory of excitation-contraction coupling"). All prior studies on Ca²⁺ sparks, however, have been performed in single enzymatically dissociated heart cells under nonphysiological conditions. In order to understand the possible significance of Ca²⁺ sparks to normal working cardiac muscle, we used confocal microscopy to record Ca²⁺ sparks, which are spatially averaged [Ca²⁺]_i transients (and Ca²⁺ waves), in individual cells of intact rat right ventricular trabeculae (composed of <15 cells in cross section) microinjected with the Ca²⁺ indicator fluo 3 under physiological conditions ([Ca²⁺]_o, 1 mmol/L; temperature, 33±1°C). Twitch force was recorded simultaneously. When stretched to optimal length (sarcomere length, 2.2 µm) and stimulated at 0.2 Hz, the trabeculae generated 700 µg of force per cell. Spatially averaged [Ca²⁺]_i transients recorded from individual cells within a trabecula were similar to those recorded previously from single cells. The amplitude distribution of the peak ratio of Ca²⁺ sparks was bimodal, with maxima at ratios of 1.8±0.3 and 2.7±0.2 (mean±SD), respectively. The amplitude of the peak of Ca²⁺ sparks was 170 nmol/L. Ca²⁺ sparks occurred at a frequency of 12.0±0.8/s (mean±SEM) in line scans covering 94 sarcomeres. Ca²⁺ waves occurred randomly at a frequency of 0.57±0.08/s and propagated with a velocity of 29.5±1.7 µm/s. The extent of Ca²⁺ wave propagation was 3.9±0.3 sarcomere lengths (sarcomere length, 2.2 µm). Ca²⁺ sparks could be identified along the leading edge of the waves at intervals of 1.30±0.11 sarcomere length. Our observations suggest that (1) Ca²⁺ sparks, similar to those recorded in single cells, occur in trabeculae under physiological conditions and (2) coupling of Ca²⁺ spark generation between neighboring sites occurs and may lead to (3) the development of Ca²⁺ waves, which propagate under physiological conditions at a low velocity over limited distances. The results suggest that concepts of excitation-contraction coupling recently derived from isolated myocytes are applicable to intact cardiac trabeculae.

Key Words: heart • excitation-contraction coupling • trabeculae • Ca²⁺ spark • Ca²⁺ wave

	Introduction

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Spontaneous release of Ca²⁺ from the sarcoplasmic reticulum (SR) is evident in single enzymatically isolated cardiac cells as spatially localized increases in [Ca²⁺]_i, termed Ca²⁺ "sparks."¹ Ca²⁺ sparks are also triggered during voltage-clamp pulses² and during action potentials,³ in which cases they have been termed "evoked Ca²⁺ sparks" or "local [Ca²⁺] transients." Recently, it has been shown that local [Ca²⁺] transients or evoked Ca²⁺ sparks are probably triggered by Ca²⁺ entering via single L-type Ca²⁺ channels.^{4 5} Ca²⁺ sparks may also trigger each other to produce Ca²⁺ waves, which propagate through the cell.⁶ It now seems unlikely that Ca²⁺ sparks arise from the opening of a single ryanodine receptor,¹ because smaller SR Ca²⁺ release events can be observed under some conditions^{7 8} and because Ca²⁺ sparks can sometimes be observed directly to have multiple sites of origin,⁹ in transverse planes at the "z" lines of the cell. Whatever their fundamental nature, however, Ca²⁺ sparks evoked by L-type Ca²⁺ currents are believed to summate, spatially and temporally, constituting the electrically evoked whole-cell [Ca²⁺]_i transient^{2 3 4 5 10} that couples excitation to contraction.

The relevance of the data on Ca²⁺ sparks to normal excitation-contraction coupling¹¹ has been largely unknown, however, because all the studies (cited above) were conducted on slack, single, enzymatically dissociated cardiac cells at room temperature, sometimes with Cs⁺ replacing K⁺ internally and externally, and often in the presence of Ca²⁺ channel blockers. Before this report, local [Ca²⁺]_i transients or spontaneous Ca²⁺ sparks had not been observed in working multicellular preparations of heart muscle under "physiological" conditions. Therefore, a major purpose of the present study was to establish whether these new concepts of excitation-contraction coupling, particularly the phenomena of Ca²⁺ sparks, are applicable to the function of working myocardium under physiological conditions. We imaged [Ca²⁺]_i with confocal microscopy of ventricular trabeculae, under physiological conditions (temperature of >30°C, pH-buffered with HCO₃/CO₂, classic physiological salines, without intracellular perfusion, without ion channel–blocking agents, and with control of sarcomere length and force production). We used rat cardiac trabeculae because they are thin enough to be metabolically stable in Krebs-Henseleit solution without perfusion of the vascular bed; thus, we could avoid possible effects of edema occurring in isolated tissue perfused with a crystalloid solution.

Similar to the situation with Ca²⁺ sparks, Ca²⁺ waves had been recorded previously only in single isolated cells,^{12 13 14} although waves of sarcomere shortening, limited to single cells, had been reported as had fast propagating waves of contraction in muscles with focal damage.¹⁵ A possibly related phenomenon, scattered light intensity fluctuations (SLIFs), had been recorded in ventricular muscle¹⁶ but had been related only indirectly to fluctuations in [Ca²⁺]_i. These phenomena have important implications for both normal and abnormal heart function; therefore, a second major purpose was to determine, in particular, whether Ca²⁺ waves of the type observed in single cells also occur in rat ventricles under physiological conditions.

The experiments required that we use confocal microscopic imaging in multicellular cardiac tissues, because spatially localized [Ca²⁺]_i transients, such as subcellular Ca²⁺ sparks or Ca²⁺ waves, cannot be observed without a method for high-resolution optical "sectioning." Since this had not been done before in multicellular cardiac muscle preparations, we have evaluated the methodology for optical sectioning of trabeculae in the present study.

	Materials and Methods

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Preparation of Trabeculae
Trabeculae from the atrioventricular border of the right ventricle of normal brown rats (LBN-F1) were obtained as described originally by ter Keurs et al.¹⁷ The trabeculae selected for study were approximately 2000 µm in length, <150 µm wide, and 60 to 75 µm thick. After isolation, the muscles were placed in a recording chamber and perfused with a physiological salt–containing solution composed of (mmol/L) NaCl 120, KCL 5, MgCl₂ 1.2, CaCl₂ 1.0, NaH₂PO₄ 2.0, Na₂SO₄ 1.2, NaHCO₃ 27.0, and glucose 10, gassed with 95% O₂/5% CO₂ (pH 7.4 with NaOH; bath temperature, 33±1°C).

Trabeculae had to be mounted in the chamber in such a way that the region to be studied was as close as possible to the bottom of the chamber, which was a glass coverslip (170 µm in thickness). The trabeculae were suspended in the recording chamber between a force transducer (type AE801, Mikro-Elektronikk) and via the tricuspid valve to a length-adjustment device (micromanipulator). The semiconductor strain-gauge force transducer was also mounted on a micromanipulator. An additional micromanipulator was sometimes used to manipulate a microtool that pushed the central region of the trabecula down against the coverslip. This served to optimize conditions for confocal visualization and to partially immobilize the muscle in the region being observed. The muscles were stimulated by pulses delivered through platinum wires affixed to the sides of the bath, parallel to the long axis of the muscle, arranged so as to achieve as uniform an activation of the muscle as possible. Muscles were stimulated at 0.2 Hz with pulses of 3-millisecond duration and 20% above threshold. Muscle length was set so that passive force was 5% of peak twitch force. Sarcomere length was 2.2 µm. Muscles were allowed to recover under these standard conditions for sufficient time (30 to 45 minutes) to develop normal function. None of the muscles showed aftercontractions, spontaneous twitches, or triggered arrhythmias.

Confocal Imaging: Optical Sectioning of Trabeculae
The confocal microscope consisted of a Bio-Rad MRC 600 imaging system connected to a Nikon Diaphot microscope. The essential problem in confocal imaging of fluorescent molecules deep within living tissues with conventional oil-immersion objective lenses is that of spherical aberration caused by mismatch of the refractive index (h) of the lens, immersion oil, coverslip (all with h=1.515), aqueous media (h=1.33), and tissue (h=1.34). In fact, the oil-immersion objective lenses used most often today (eg, Nikon; numerical aperture, 1.4; 60x oil-immersion plan-apochromat) lose intensity and resolution at depths >10 µm into an aqueous solution,¹⁸ making them suitable mainly for use only with single isolated cells. It has been recognized recently that the solution to this problem is in the use of new "water immersion" objective lenses with variable optical correction for a glass coverslip. These objective lenses are designed for use with aqueous specimens and immersion medium (both of h=1.33) and can be "corrected" for the unavoidable refractive index mismatch produced by the use of a glass coverslip (h=1.51). Accordingly, our images were obtained with the Nikon CFN plan-apochromat x60 water immersion objective (numerical aperture, 1.2). This objective has a working distance of 220 µm (>170 µm of coverslip glass). To facilitate optical sectioning, which was required to evaluate the quality of the images, we modified the confocal microscope with a piezoelectric focusing device for obtaining fast and reliable positioning of the objective lens. Images were analyzed using IDL (Research Systems, Inc) on an IBM RS/6000 workstation (IBM Corp).

Loading Trabeculae with Fluo 3 Salt Versus Fluo 3-AM
We evaluated the efficacy and relative merits of two methods of loading the trabeculae with fluo 3. Some trabeculae were loaded with fluo 3 by exposure to fluo 3-AM (50 µmol/L for 45 minutes, room temperature). Other trabeculae were loaded with fluo 3 by iontophoresis¹⁹ of fluo 3 (salt) after impalement with a microelectrode (250 to 300 M when filled with 1 mmol/L fluo 3). In these muscles, resting membrane potentials were typically -75 mV. Hyperpolarizing iontophoretic current of 5 to 8 nA was applied for 10 to 20 minutes. The major differences in the appearance of trabeculae loaded by the two methods were that (1) in muscles loaded by exposure to fluo 3-AM, cells other than muscle cells were evident, and (2) the muscle cells were loaded uniformly, but very lightly (relative to cells that had been microinjected). In general, it was less difficult to load trabeculae with fluo 3 to usable concentrations by microinjection than by exposure to fluo 3-AM. One reason may be that the cells of the epicardial layer present a barrier to the transport of fluo 3-AM into the interior of the trabecula. We note that when this barrier surrounding the trabecula is considered, the surface-to-volume ratio of a trabecula is small compared with that of a single cell, so that the rate at which fluo 3-AM reaches muscle cells in a trabecula may be relatively small. On the other hand, it is apparent (eg, see Fig 1) that loading fluo 3 (salt) by iontophoresis from a microelectrode does not load all the cells uniformly. It is probable that fluo 3 binds to molecules (perhaps fixed) within the cell,²⁰ and fluo 3 must diffuse from cell to cell through gap junctions. Fluo 3 has a relatively high molecular weight of 960, so it is likely that both factors hinder diffusion from cell to cell. We routinely used iontophoresis, because with this technique higher concentrations of fluo 3 were achieved, and loading of fluo 3 into mitochondria and nonmuscle cells was avoided.

View larger version (102K): [in this window] [in a new window]   Figure 1. Optical sectioning of rat ventricular trabecula microinjected with fluo 3. Each image is the average of 10 frames in the "slow-scan" mode of the confocal microscope (MRC-600, Bio-Rad). The images were taken at intervals of 4.0 µm in the z axis, starting near the bottom (upper left panel) and moving upward (left to right, top to bottom). Image at lower right is thus 48 µm into the trabecula.

	Results

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Optical Sectioning of Trabeculae
First, we evaluated the performance of the system in imaging fluo 3 fluorescence arising from cells deep within a trabecula. Results are presented in Fig 1, where, from left to right and top to bottom, the images were taken at intervals in the z axis of 4.0 µm through a trabecula that had been microinjected with fluo 3 (salt). In this case, the trabecula was resting on the bottom of the chamber (glass coverslip). The image at bottom right is thus at a depth of 50 µm into the preparation. Clearly, confocal "optical sectioning" of the trabecula has been achieved. For example, although nuclei of cardiac cells often contain more fluo 3 than does the surrounding cytoplasm, they are often not visible at all in conventional epifluorescence microscopy of single cells, because they are obscured by "out-of-focus" fluorescence. In these confocal images, however, nuclei of individual cells are clearly visible and are "brightest" in just one section. They disappear within two sections, as the plane of focus is moved upward, consistent with rod-shaped structures having a diameter of 8 µm^{21 22} and the point-spread function of a confocal microscope.²³

Ca²⁺ Sparks, Ca²⁺ Twitch Transients, and Ca²⁺ Waves in Ventricular Trabeculae
Confocal imaging was performed in microscopically quiescent trabeculae, as illustrated in Fig 2A through 2C. Either full-frame images (x, y, and constant z) (Fig 2A) or "line-scan" images [x(t), constant y, constant z] (Fig 2B) were obtained. Ca²⁺ sparks are readily visible in the full-frame image as spatially localized bright regions and in the line-scan images as localized transient changes in fluorescence. In line-scan images, Ca²⁺ waves were apparent as regions of elevated [Ca²⁺]_i that moved at constant velocity (see Fig 4). During the 30 minutes that this muscle was studied, it was microscopically quiescent unless stimulated. When it was stimulated at 0.2 Hz (Fig 2D), it developed 10 mg of force at the peak of the isometric twitch contraction. The spatially averaged [Ca²⁺]_i transient, obtained by integrating line-scan images during stimulation, was similar to that recorded previously with a similar technique in single cells² and to the spatially averaged [Ca²⁺]_i transient recorded with conventional fluorescence microscopy of single rat cells. (Line-scan images during contraction are not presented here, however, because internal shortening interfered with their interpretation.) The peak isometric twitch force corresponded to 700 µg per cell, since this trabecula was composed of 15 cells in cross section. Peak stress development was thus near 70 mN/mm² as reported previously for muscles in a healthy physiological condition.^{24 25}

View larger version (62K): [in this window] [in a new window]   Figure 2. Ca2+ sparks during prolonged quiescence in a normal rat ventricular trabecula. The figure illustrates the fact that Ca2+ sparks occur spontaneously in rat trabeculae, when quiescent under physiological conditions, and that the same trabeculae are capable of generating normal contractions and [Ca2+]i transients when stimulated. A, Full-frame image during quiescence. B, Line-scan image, along line indicated in panel A. C, Line plots derived from line-scan image in panel B. In panel A, the solid white vertically oriented line through the image indicates the single line that was scanned 512 times at a rate of 500 Hz to produce the line-scan image. Note the length calibration in upper left. In panel B, the line-scan image presents fluorescence along the scan line as a function of time (horizontal bar indicates 200 milliseconds). Vertical calibration is the same as in panel A. In the line-scan image, spontaneous Ca2+ sparks are evident as the transient spatially localized increases in fluorescence. In panel C, line plots (fluorescence as a function of time) at eight different locations along the scan line, indicated by the numbers and dashed white arrows, are shown. The line plots were obtained by averaging the fluorescence from 5 pixels (1.36 mm) at the place indicated by the tips of the arrows. D, Contraction and spatially averaged [Ca2+]i transient. Fluor. indicates fluorescence. The numbers in panel B correspond to the numbers of the line plots shown in panel C.

View larger version (87K): [in this window] [in a new window]   Figure 4. Ca2+ waves. The propagation of different Ca2+ waves was different, even when generated at the same or nearby sites in a cell. In panel A, the Ca2+ wave has an asymmetrical appearance, as if it encountered a border or failed to propagate in one direction. In panel B, the wave begins as a "V," indicating equal propagation in both directions, but then stops propagating, even in a region through which a previous wave (panel A) had propagated. The black arrows in panels A and B mark the same position in the two scans, indicating that the two waves started at the same place. The white arrows indicate the positions of sparks at the leading edge of the wave in panel A.

Ca²⁺ Sparks
Ca²⁺ sparks occurred at an average rate of 11.92±0.84 per line-scan image, with 11% of these being generated from repeatedly firing single sites. Line plots showing the time course of fluorescence in selected 1.36-µm portions of the scan line (5 pixels) are shown in Fig 2C. In unstimulated muscle (Fig 2A through 2C), Ca²⁺ sparks were similar in time course and spatial spread to those recorded previously in single isolated cardiac cells. As we have pointed out previously,²⁶ a number of factors shape the confocal image of Ca²⁺ sparks. Variation in amplitude and time course can be seen in Fig 2C even in Ca²⁺ sparks that occurred as a result of repetitive "firing" in a single site (Fig 2C, site 6). The observation in the latter site shows that the second of two successive Ca²⁺ sparks was larger than the first one, suggesting that rapid fluctuations of the Ca²⁺ load of the SR were not the cause of variation of Ca²⁺ spark fluorescence.

The images presented in Fig 3A are the average of 79 single Ca²⁺ sparks and are aligned with respect to peak amplitudes, where the spatial distribution and time course of the fluorescence ratio are displayed as the extent and height of a surface (upper wire-frame diagram) and with color-coding (below) and were calculated according to the methods we used previously in single isolated cells. The fluorescence ratio was calculated by assuming that the lowest fluorescence in the image at the site of the Ca²⁺ spark represented fluorescence at the normal "resting" [Ca²⁺] (100 nmol/L). The time to rise from 10% to 90% of the peak was 5 milliseconds, and the time to fall from peak to half of peak was 40 milliseconds (Fig 3A and 3C). Since the Ca²⁺ sparks used for the average were spatially restricted to regions <2 µm, the average may represent an "in-focus" Ca²⁺ spark in these trabeculae, under nearly physiological conditions. It was striking that the average Ca²⁺ spark was preceded by a rise of the [Ca²⁺], which started 10 milliseconds before the Ca²⁺ spark (Fig 3C). Such an event, which suggests the possibility of triggering of some of the Ca²⁺ sparks by a local rise of [Ca²⁺]_i, has been described before in isolated cells.⁸ The width of the region with elevated Ca²⁺ increased (Fig 3B) after the peak of the Ca²⁺ sparks, consistent with diffusion of Ca²⁺ away from the Ca²⁺ spark–generating site, as has been shown in isolated cells.⁹ In Fig 3D, we present the properties of all Ca²⁺ sparks, as measured in a single muscle. The distribution of peak fluorescence was broad (not shown). When these Ca²⁺ sparks were converted to fluorescence ratios, the distribution appeared to be bimodal, with a large population that could be fit reasonably well by a single gaussian distribution and a second, very small, population of larger Ca²⁺ sparks (Fig 4B). A similar distribution has been found in single isolated cells. Ca²⁺ sparks did not occur randomly in the muscle, as shown in Fig 3E. Between the apparent cell boundaries, the average spacing between sites at which Ca²⁺ sparks occurred was 2 µm, consistent with the sarcomere length and previous reports. Scanning was perfectly longitudinal in these muscles because of the way that they were oriented in the recording chamber, so it was not possible to determine whether some Ca²⁺ sparks had multiple sites of origin in the transverse plane, as reported in single cells.⁹ Some cells generated Ca²⁺ sparks at a low frequency, and some sites were much more frequent than others, within a given cell (peaks in Fig 3E).

View larger version (29K): [in this window] [in a new window]   Figure 3. Properties of Ca2+ sparks in ventricular trabecula. A, Average of 97 selected Ca2+ sparks. B, Normalized spatial profiles of fluorescence ratio at peak (black), 22 milliseconds (red), 52 milliseconds (green), and 124 milliseconds (blue). C, Time course of average Ca2+ spark, taken through the center or site of origin. D, Population histogram of peak fluorescence ratios of sparks in one muscle. Gaussian distributions are as follows: centers, 1.9 and 2.8; widths, 0.45 and 0.36; heights, 25.29 and 2.4; and areas, 18.0 and 1.4. E, Distribution of Ca2+ spark sites during 25 seconds of scanning. Some sites generated sparks more frequently than expected for a strictly stochastic process.

In addition to single Ca²⁺ sparks, the line scans showed that Ca²⁺ sparks larger in extent than 2 µm occurred at 8% of the frequency of the single Ca²⁺ sparks. The frequency of occurrence of these >2-µm Ca²⁺ sparks was slightly higher than the frequency that we have reported for single cells and was smaller than the frequency of generation of Ca²⁺ sparks in repetitively firing sites (30% of all small Ca²⁺ sparks).

Ca²⁺ Waves
We did not observe any Ca²⁺ waves in groups of cells propagating along the muscles such as has been described in muscle with foci of damage,¹⁵ but slowly traveling Ca²⁺ waves were recorded in some of these trabeculae (Fig 4). When they occurred, they did so several seconds after the twitch, at a time when inspection through the microscope revealed occasional shortening of small populations of sarcomeres, which traveled as waves through individual cells. In general, the Ca²⁺ waves recorded in these preparations appear comparable to those we have recorded previously in single cells.^{12 13 14} Because it takes several seconds to store a single image from the MRC-600 confocal microscope, we have been unable so far to evaluate the velocity of the waves from images in the full-frame mode. However, in line-scan images, Ca²⁺ waves could be observed unequivocally, as a region of elevated [Ca²⁺]_i that moved linearly with time (no exceptions to this were found in any of the line scans). Ca²⁺ waves occurred rather rarely in these trabeculae at the [Ca²⁺]_i and temperature used in the present study. Their average frequency in 64 line-scan images encompassing 94 sarcomeres was 0.57±0.09 Hz, and the extent of Ca²⁺ waves was, on average, 3.9 sarcomere lengths (see the Table). The frequency of waves per cell may be estimated from these observations as follows: The extent of the Ca²⁺ waves was, on average, 4 sarcomere lengths. Thus, at a sarcomere length of 2.0 µm, the Ca²⁺ waves probably extended 8 µm in all directions. Consequently, 2200 µm³ of the cell was involved in a wave or 1/9 of the cell volume. This gives a frequency of 9x0.57/2 Ca²⁺ waves per cell, or 2.5 Hz. This may be a slight underestimate, because we did not qualify Ca²⁺ gradients that extended over less than three sarcomeres as Ca²⁺ waves. More often than not, these waves had an asymmetric appearance such as one would expect when they are propagated in only one direction. One cause for the asymmetric appearance might be that they started at an edge of a cell. On the other hand, Fig 4 shows that of two waves that started at the same site in the cell (horizontal black arrows), one propagated in only one direction (panel A), whereas the other propagated in both directions (panel B). This suggests that if these waves started at a gap junction, their propagation into one or both cells connected to the gap junction was dictated by chance.

View this table: [in this window] [in a new window]   Table 1. Properties/Values of Ca2+ Sparks and Ca2+ Waves

The Table shows that the distance over which the Ca²⁺ waves propagated was small. One reason that the Ca²⁺ waves may have stopped after such short propagation distances is that they may have originated close to the border of cells and stopped at the end of the cell. However, it is unlikely that all of the Ca²⁺ waves that we have encountered were initiated near the end of cells and therefore stopped by the cell border. Also, Fig 4 shows that of two Ca²⁺ waves that started in the same region of the cell, one Ca²⁺ wave stopped earlier than the other Ca²⁺ wave, so that it is unlikely that both Ca²⁺ waves were stopped by a cell border. Last, nearly all Ca²⁺ waves terminated with a decline and spread similar to those of the Ca²⁺ sparks, whereas termination at the cell edge should have been reflected by an acute border of the Ca²⁺ wave. Therefore, we consider it more likely that these Ca²⁺ waves both propagated slowly (29 µm/s) and terminated spontaneously because the propagation process was not reliable under the conditions of these experiments; ie, the safety factor for propagation was low. The same low safety factor for propagation may have caused Ca²⁺ waves to propagate often only in one direction. The low frequency of the Ca²⁺ waves in the line scans was in agreement with the sporadic random occurrence of waves of shortening of small groups of sarcomeres that we observed using direct microscopy. It was clear that the waves of sarcomere shortening also propagated over distances less than one cell length, as has been described previously.¹⁴

It was striking that nearly all Ca²⁺ waves showed Ca²⁺ sparks on their leading edge (see the Table), with an average distance of Ca²⁺ sparks along the edge of 1.3 sarcomere lengths. The Ca²⁺ sparks along the edge of the Ca²⁺ waves could not always clearly be identified, possibly because of the out-of-focus position of the Ca²⁺ spark–generating site. This may suggest that Ca²⁺ sparks may have been present at the edge of the Ca²⁺ waves at intervals of 1 sarcomere length, which would be similar to the conclusion, reported by Cheng et al,⁶ that in single myocytes Ca²⁺ sparks may provide the regenerative mechanism for a Ca²⁺ propagation wave from terminal cisterna to terminal cisterna.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown, for the first time, that [Ca²⁺]_i can be visualized with confocal microscopy in individual muscle cells within a ventricular trabecula under physiological conditions, including mechanical loading and force generation. Through the use of an appropriate objective lens, adequate spatial resolution of subcellular structures can be obtained, even deep within trabeculae.

The present results show that spontaneous Ca²⁺ sparks are common in unstimulated quiescent rat ventricular trabeculae. We estimated that the frequency of Ca²⁺ sparks per cell is 330 Hz, ie, a slightly higher value than that reported for isolated cells. The difference may be due to the difference in temperature between the present study (34°C) versus the temperature at which studies on single cells are commonly conducted.^{1 2 3 4 5} Another reason may be that these trabeculae were taken from the right ventricle, in contrast to the majority of the myocytes isolated randomly from the heart.

The amplitude of the fluorescence of the Ca²⁺ sparks varied 5-fold; the peak ratio of the Ca²⁺ sparks varied 2-fold. The distribution of spark amplitudes was similar to the one that has been reported before for isolated myocytes from normal myocardium.²⁷ Fig 3D shows that the distribution could be fitted by a bimodal distribution with gaussian distribution around maxima of peak ratios at 1.8±0.3 (mean±SD) and 2.7±0.2 (suggesting a 2-fold difference in the peak amplitude of the sparks [197 and 393 nmol/L, respectively] at a diastolic [Ca²⁺]_i of 90 nmol/L). We favor the interpretation that the distribution was bimodal rather than the possibility that the distribution was skewed. In general, frequency distributions of image parameters, such as peak Ca²⁺ spark amplitude or rise time, are not expected to be gaussian or even unimodal, even if the underlying events are all exactly the same. This is true, because the confocal microscope still detects some fluorescence from Ca²⁺ sparks that arise outside the plane of focus and because Ca²⁺ released from sites outside the plane of focus diffuses into the region being observed. Ca²⁺ sparks arising outside the region being scanned (ie, out of focus) are expected from theory to be broader at the peak and smaller in peak amplitude and to rise and fall more slowly. Finally, since Ca²⁺ sparks arise mainly at transverse tubules,²⁸ the observed spark parameters may segregate into groups, reflecting the small number of sites at which Ca²⁺ sparks can originate and be detected, given a particular scan line. A possible cause for the slightly skewed distribution of Ca²⁺ spark ratios is that the ratio was calculated from the peak amplitude of the Ca²⁺ spark divided by the lowest fluorescence at the same site during the line scan. Hence, if the peak [Ca²⁺]_i during the Ca²⁺ sparks depended on the diastolic [Ca²⁺]_i, one would have expected a change in the distribution of ratio amplitudes, with a skewing favoring the occurrence of a particular population of Ca²⁺ sparks. This possibility seems less likely to us because the amplitude of the sparks was only slightly dependent on the diastolic [Ca²⁺]_i.

The Ca²⁺ sparks observed here reached a peak amplitude of 170 nmol/L, which is below the level at which crossbridges are activated in intact trabeculae. Furthermore, the Ca²⁺ sparks are spatially restricted, suggesting that the [Ca²⁺] in the myofilament space during and after the peak of the Ca²⁺ spark must have been substantially lower than 170 nmol/L, which makes it even more unlikely that crossbridges are activated by individual Ca²⁺ sparks. The observation that Ca²⁺ sparks occur in microscopically quiescent muscles is therefore not surprising.

Ca²⁺ waves occurred at a 20-fold lower frequency than Ca²⁺ sparks (Table) and propagated slowly over short distances in single cells. In the above, we have argued that these properties were probably caused by a low safety factor for propagation; ie, the probability of starting and maintaining a propagating Ca²⁺ wave was low under the conditions of our experiments.

Our observation that nearly all Ca²⁺ waves showed Ca²⁺ sparks on their leading edge is similar to the previous observation in single myocytes by Cheng et al⁶ and suggests that Ca²⁺ sparks may provide the regenerative mechanism for wave propagation and that diffusion from terminal cisterna to terminal cisterna would form the conduction mechanism. In this case, the trigger for Ca²⁺ spark generation during a propagated wave would consist of Ca²⁺ arriving from an adjacent Ca²⁺ spark–generating site.⁶ If Ca²⁺ release from the site is proportional to the rate of rise and the absolute [Ca²⁺]_i reached at the Ca²⁺ spark site, one would anticipate that the waves propagate at a constant velocity, because the same process would repeat itself at each following site.

Neither multicellular waves of Ca²⁺ nor multicellular waves of sarcomere shortening (nor triggered arrhythmias) were observed in these muscles. The Ca²⁺ waves seemed to occur randomly (ie, not at a focus of damage), and their presence was correlated with microscopically visible waves of contraction of small groups of sarcomeres that traveled as waves within individual cells in the muscle. [Ca²⁺]_i waves and propagating contractions occurred, and they did so several hundred milliseconds after the twitch, ie, usually later than the start of occurrence of the Ca²⁺ sparks. These results may provide an explanation for spontaneous motion observed previously in the form of SLIFs in rat papillary muscles.^{16 29} In this regard, it has been reported recently that the frequency of Ca²⁺ sparks recovers after stimulation, similar to that observed for SLIFs.²⁹ If single Ca²⁺ sparks precede the development of Ca²⁺ waves, it is conceivable that SLIF accompanies Ca²⁺ waves and that Ca²⁺ sparks remain undetected by techniques that monitor sarcomere motion.

Finally, the existence of Ca²⁺ sparks in muscles generating normal [Ca²⁺]_i transients and twitch force implies that modern concepts suggesting an essential role of Ca²⁺ spark generation in excitation-contraction coupling apply to intact normal cardiac muscle, just as they do to single isolated cells under voltage-clamp and internal perfusion.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-29473 (Dr Wier) and HL-02466 and HL-50435 (Dr Balke); an American Heart Association, Maryland Affiliate, Inc, Grant-in-Aid (Dr Balke); and a grant from the Division of Cardiology, Department of Medicine, University of Maryland at Baltimore. National Institutes of Health grant HL-44065 (Dr Marban) supported Dr Gao and the microinjection of fluo-3 into trabeculae that was performed at The Johns Hopkins University. Dr ter Keurs holds a Medical Scientist position of the Alberta Heritage Foundation for Medical Research; his research is supported by the Alberta Heart and Stroke Foundation.

Received March 31, 1997; accepted June 26, 1997.

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