Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers

Murali K. D. Pagala1 and Stuart R. Taylor2

1 Neuromuscular Research Laboratory, Maimonides Medical Center, Brooklyn, New York 11219-2999; and 2 Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905-0001

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fast-twitch and slow-twitch rat skeletal muscles produce dissimilar contractures with caffeine. We used digital imaging microscopy to monitor Ca2+ (with fluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes in Ca2+ in individual fibers were markedly different from average responses of a population. All fibers showed discrete, nonpropagated, local Ca2+ transients occurring randomly in spots about one sarcomere apart. Caffeine increased local Ca2+ transients and sarcomere motion initially at 4 mM in soleus and 8 mM in extensor digitorum longus (EDL; ~23°C). Ca2+ release subsequently adapted or inactivated; this was surmounted by higher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction in both types of fiber. In EDL fibers, 16 mM caffeine moderately increased local Ca2+ transients. In soleus fibers, 16 mM caffeine greatly increased Ca2+ release and produced propagated waves of Ca2+ (~1.5-2.5 µm/s). Ca2+ waves in slow-twitch fibers reflect the caffeine-sensitive mechanism of Ca2+-induced Ca2+ release. Fast-twitch fibers possibly lack this mechanism, which could account for their lower sensitivity to caffeine.

dissimilar caffeine sensitivity; calcium release from sarcoplasmic reticulum; parvalbumin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CAFFEINE HAS BEEN USED widely to bypass voltage-gated excitation-contraction coupling in skeletal muscle, directly gate Ca2+-release channels in the sarcoplasmic reticulum (SR), and generate Ca2+-activated contractures. The features of a caffeine contracture differentiate slow-twitch mammalian skeletal muscles from less caffeine-sensitive fast-twitch muscles (9, 13, 22, 31). The aforementioned studies relied solely on the dimensions of isometric force developed by stretched muscles. They assumed that the rate and amplitude of SR Ca2+ release are directly related to force. However, caffeine has multiple effects on muscle (32-34). Which effects account for the differential caffeine sensitivity is uncertain.

Low doses of caffeine produce motion in individual sarcomeres (10, 11, 14). Low doses of caffeine also increase submaximal Ca2+-activated force in stretched skinned fibers, and large doses of caffeine depress maximum Ca2+-activated force and depress the activity of several enzymatic reactions (26, 38). Caffeine potentiates the mechanism of Ca2+-induced Ca2+ release (CICR), if the Ca2+ concentration in the SR is measured by the isometric force resulting from the application of a high concentration of caffeine (5). The total Ca2+ content of the SR in skinned fibers has also been measured by equilibrating fibers with Ca2+ buffers, followed by lysis. This technique shows that the SR of fast-twitch fibers is only one-third full at the myoplasmic Ca2+ concentration of a resting fiber, whereas the SR of slow-twitch fibers is saturated with Ca2+ (8). However, results using skinned fiber tension development to monitor Ca2+ release have not uniformly confirmed the assumption that force reflects Ca2+ release. Some investigators have concluded that Ca2+ release from slow-twitch fiber SR is more sensitive to caffeine than Ca2+ release from fast-twitch fiber SR, whereas others have concluded that the Ca2+ sensitivity of the myofilaments is the critical factor, rather than Ca2+ release (30, 36).

Many studies of Ca2+-release channels (ryanodine receptors) have been performed on preparations of SR removed from muscle. The sensitivity of these preparations is uniquely affected by the conditions chosen to mimic the intracellular environment, which might account for some conflicting conclusions (19, 23). Studies of isolated SR vesicles and SR Ca2+-release channels in lipid bilayers show that the channels are activated by Ca2+ as well as by caffeine, and the sensitivity of fast SR channels to caffeine is similar to that of slow SR (19, 23, 29). There is no correlation between fiber type and ryanodine binding to isolated SR vesicles, leading some to conclude that the contractile properties of fast-twitch and slow-twitch muscles are not due to differences in their ryanodine receptors (6). Until recently, most investigators focused on channel properties measured under constant conditions. Time-resolved studies of skeletal ryanodine receptors in bilayers now show that Ca2+ activation of ryanodine receptors is followed by their adaptation or inactivation at constant Ca2+ concentration. Ryanodine receptors may be regulated by Ca2+-dependent activation and Ca2+-dependent inhibition mechanisms that gate independently (18).

The first systematic study of caffeine effects on Ca2+ in wholly intact mammalian skeletal muscle measured changes in Ca2+ from average responses of populations of fibers. The results led to the suggestions that fast-twitch SR Ca2+-release channels are less sensitive to caffeine than those of slow-twitch fibers and that parvalbumin in fast-twitch muscles may buffer Ca2+ released by low concentrations of caffeine and prevent any useful rise in Ca2+ until high concentrations are applied (7).

To eliminate possible differences in SR function caused by its isolation from a natural environment and to detect possible differences between individual cells and a population, we studied wholly intact rat muscle fibers, with the milieu of the SR Ca2+ channels and contractile proteins determined by the intrinsic competence of the cells themselves. We used digital imaging microscopy to measure the motion of sarcomeres in fibers dissected from fast-twitch and slow-twitch rat skeletal muscles and used a fluorescent probe to measure the spatial and frequency domains of discrete, local Ca2+ transients. Our results show that Ca2+ transients from a population of cells obscure the large variation in local Ca2+ transients among individual cells. Furthermore, we found that the CICR mechanism can be activated by caffeine in slow-twitch fibers only and that the CICR gives rise to propagating waves of Ca2+.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rat muscles and fiber dissection. The data were obtained from five soleus and five extensor digitorum longus (EDL) muscles isolated from five anesthetized male Sprague-Dawley rats weighing ~300 g. Data from fibers that moved from the field of view during an experiment were omitted. The muscles were dissected under dark-field illumination down to thin sheets of intact fiber bundles (1 mm or less in diameter and 0.1-0.3 mm thick). The fibers were stimulated with brief pulses (~5 V and 1 ms) during and after dissection to assure that they were able to develop propagated contractions. Data collected from 28 soleus fibers and 40 EDL fibers were selected for this report. The selection of fibers with either very few or a great many light-scattering particles produced bundles of soleus and EDL fibers that evidently were essentially type I or type II, respectively (31). A very small portion of rat EDL fibers are slow twitch (marked by an antibody that recognizes the slow class of myosin heavy-chain isoforms), but these slow-twitch fibers are concentrated in the medial portion of the EDL and are absent from the lateral portion that we used for this study (1). Previous measurements of dense staining for ATPase made by one of us (M. K. D. Pagala) confirm the classification of rat fibers dissected in this manner (31).

Experimental solutions. Physiological solutions were composed of (in mM) 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 1 Na2HPO4, 15 NaHCO3, and 11 glucose. The solutions were bubbled continuously with 95% O2 and 5% CO2. Caffeine was added as the free base to achieve the final concentrations noted. All experiments were conducted at ~23°C. The chamber was continuously perfused with solution. The input tube was directly over the fibers in the field of view, and the delay between switching solutions and the arrival of new solution at the surface of the cells was <3 s. The solution was perfused across the surface of the cells in a direction 90° from the long axis of the fibers at a speed of ~500 µl/s. The purpose was to minimize or eliminate effects of the partially characterized factor secreted by skeletal muscle cells incubated in caffeine (12, 14). At the end of a series of four incremental increases in caffeine (4, 8, 16, and 32 mM), the fibers were exposed to 150 mM KCL and isosmotic CaCl2 buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. At the end of two experiments an organic lipid solvent was added as well. These terminal solutions produced a rise in fluorescence that equaled the previous increase caused by 16 mM caffeine (2.4% greater on average; n = 6). This indicated that the lack of effect of the final dose of caffeine (32 mM) was not the result of a change in the dye (e.g., bleaching, compartmentalization) or dye washout from the cells.

Imaging Ca2+. The microscope was a Zeiss IM-35 fitted with an all-quartz pathway and a Nikon quartz, ultraviolet-transmitting objective (×20, numerical aperture 0.80), a Zeiss 150-W xenon lamp, and excitation and emission filters from Molecular Probes. One port of the microscope was connected to a Spectra Sources MCD-220 camera. The field of view was a 113 × 113-µm planar optical section about four to eight fibers wide.

The cells were loaded with fluo 3-acetoxymethyl ester (AM) after the bundles were dissected and mounted on the microscope stage. Fluo 3-AM (50 µg; Molecular Probes) was dissolved in dimethyl sulfoxide (50 µl) and stored at -20°C. Loading solutions were made by mixing 9 µl fluo 3-AM solution and 1 µl of Pluronic acid (F-127) in 2 ml of normal physiological saline. After the fibers were loaded, the dye was removed from the bath by perfusing the chamber with ordinary saline. The fluorescence from fibers untreated with caffeine remained stable over many hours if we limited exposure to light with an automatic shutter (Uniblitz; Vincent Associates, Rochester, NY). The fluorescence emission intensity (>530 nm for fluo 3-AM) was determined with excitation at 488 nm. We tested for significant differences in average myoplasmic Ca2+ by applying a two-tailed test to each population mean and its SD (P < 0.05, Student's t-test).

Figures 2-4 and 6-9 (concerning Ca2+) are calibrated relative to the 12-bit/pixel range of the camera. The plots of average intensity for a population of fibers are depicted with the percent maximum pixel output on the y-axis and with time on the x-axis. The fluorescent images are color coded and labeled with calibration bars showing the range of values. Original images often appeared to be nearly identical to one another, but, if they were subtracted from their nearest neighbor in time, the results were difference images with the direction of object displacement the same as the direction of the difference (2).

Imaging sarcomere motion. The tendons were fixed after allowing the unrestrained fibers to assume their slack length. Sarcomere spacing at slack length was measured after sequential stages of processing, as previously described (28). We also measured the original spatial domain images in the frequency domain, to detect frequencies and orientations associated with motion in individual sarcomeres. Each image was transformed with fast Fourier transform algorithms, as previously described (28).

The images were acquired and analyzed with customized software (PixCell and ANALYZE, Mayo Foundation). The acquisition program simultaneously displayed the brightest 8 bits of the 12-bit/pixel dynamic range, the histogram of each image, and cursors that automatically located the brightest part of each image. The cursors, and their associated intensity values, were used to adjust focus before acquisition of the next image. Transmitted light images or low-light-level images were stored consecutively <2 s after they were acquired and displayed. The exposure times were 0.1 s for transmitted light images and 5 s for fluorescent images. We used the simultaneous display of an image and its histogram during acquisition to estimate the bright and dark areas caused by features in a cell, divided by the noise level when the cell was removed from the same field. We selected an exposure time of 5 s on the basis of this estimate and calculated the signal-to-noise ratio. The signal-to-noise ratio calculated from the peak-to-peak signal divided by the root mean square noise was 47 dB.

Two disadvantages of the long exposure time for our fluorescent images were the sacrifice of temporal resolution and the inclusion of a volume of unknown depth on the z-axis. These are not limitations when the events measured as Ca2+ indicator dye fluorescence changes occur over one plane of an image obtained by confocal microscopy (17, 35).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bright field images. We took bright field images at intervals between fluorescent images. They were processed to determine the orientation and boundaries of the fibers, movement in individual sarcomeres, and the average sarcomere spacing. The average spacing in groups of 10 contiguous striations allowed to assume their slack length was statistically the same in both EDL and soleus fibers, 1.91 ± 0.02 µm (n = 11). Slight shortening in single sarcomeres was easily detected because the fibers were slack. Figure 1 shows six consecutive difference images of EDL fibers taken in transmitted light. The fibers were exposed to 16 mM caffeine 5 min before the first of the images shown (Fig. 1, top left).


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Fig. 1.   Patterns of sarcomere motion induced by caffeine. Transmitted light difference images of rat extensor digitorum longus (EDL) fibers are shown after application of caffeine (16 mM). Images are consecutive from top left to top right and then bottom left to bottom right. Fibers are oriented 135° relative to bottom edge of each frame. Light and dark regions correspond to distance and direction of sarcomere motion.

Motion detection.. To measure motion in a difference image, we assumed that an object of constant intensity moved with uniform velocity over a background of constant intensity. Two disjoint regions were generated by the subtraction process. One region was the result of the leading edge, and the other resulted from the trailing edge. Difference images of fibers in caffeine clearly showed these features in transmitted light (Fig. 1). The fluorescent images we show (Figs. 6-9) always lacked these features. The motion of one or more sarcomeres produced symmetrical displacement of rows of striations, along the axis of individual myofibrils, in specific regions of a fiber. Some sarcomeres presumably shortened actively while others in series were dragged passively. The transforms of these images showed frequencies in the power spectra corresponding to the striation spacings and a direction corresponding to the orientation of sarcomeres in the myofibrils.

The average sarcomere spacing measured from the transmitted light images was unchanged by raising the caffeine concentration, but difference images showed that portions of individual fibers shortened and relaxed periodically over distances ranging from a single sarcomere to a string of at least 10 (Fig. 1). The long exposure time of the transmitted light images (100 ms) prevented us from determining the timing of these sarcomere events. These events presumably correspond to the caffeine-induced sarcomere oscillations studied by others (10, 11, 14, 25).

Length dependence of mechanical activity. Contractile force generated by the initial dose of caffeine (4 mM) was insufficient to appreciably move the fibers or produce sustained motion in individual sarcomeres. Stretch increases the sensitivity of skinned rat muscle fibers to activation of the myofilaments by low Ca2+ concentration (37). Apparently, the mechanical sensitivity to low concentrations of Ca2+ was also small in intact rat fibers at slack length, compared with stretched fibers (4).

Thresholds for mechanical activity. The caffeine concentrations at which the transmitted light difference images showed threshold mechanical activity differed between soleus and EDL fibers. For example, difference images of EDL fibers taken at the left asterisk in Fig. 2 showed no motion. In addition, the Fourier transforms of fluorescent images acquired after the left arrowhead on the horizontal line in Fig. 2 also showed no motion, but mechanical activity was evident in the same EDL fibers, at the right asterisk in Fig. 2. Figure 1 begins at the time indicated by the right asterisk in Fig. 2. All the soleus fibers, on the other hand, shortened slightly in 4 mM caffeine before relaxing spontaneously. Stretched fibers also relax spontaneously and only occasionally develop persistent caffeine contractures at warm temperatures (20).


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Fig. 2.   Effect of caffeine on Ca2+ in rat EDL muscle fibers. Time course of average changes in fluorescence emission from EDL muscle fibers is shown. Fibers were exposed to increasing concentrations of caffeine at times indicated (bottom). Intervals when fibers were imaged in transmitted light occurred at asterisks. Right asterisk marks start of Fig. 1. Horizontal line with arrowheads marks period depicted in Fig. 11. CCD, charge-coupled device; f, frame.

Higher concentrations of caffeine (8 and 16 mM) produced transient local shortening in two-thirds of the soleus fibers, and 32 mM caffeine produced no mechanical response in either type of fiber. Prolonged exposure to caffeine evidently depressed myofilament interaction in both types of fiber. Because large increases in caffeine concentration failed to produce any remarkable change in mechanical activity but did produce striking differences in Ca2+ release, we focused our attention on the latter.

Effects of caffeine on fluorescence from populations. Figure 2 shows the average fluorescence of a population of EDL fibers and the effects of progressively increasing the caffeine concentration. The delay between raising caffeine to 8 or 16 mM and an increase in Ca2+ in the EDL fibers was ~80-90 s in the population average.

Figure 3 shows the same experiment on soleus fibers. The delay between elevating caffeine and an increase in the average fluorescence varied between 32 and 195 s (4 mM), 20 s (8 mM), and <8 s (16 mM).


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Fig. 3.   Effect of caffeine on Ca2+ in rat soleus muscle fibers. Time course of average changes in fluorescence emission from soleus muscle fibers is shown. Fibers were exposed to increasing concentrations of caffeine at times indicated (bottom). Intervals when fibers were imaged in transmitted light occurred at asterisks.

Comparison of the collective population responses. Figure 4 shows the average changes in fluorescence from all the soleus and EDL muscle fibers. Although individual EDL fibers were clearly less sensitive to caffeine than soleus fibers, 16 mM caffeine was the only dose for which the average population difference between EDL and soleus fibers was significant (0.02 P < 0.05). Ca2+ always fell to the control value or less before the next highest dose of caffeine was introduced.


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Fig. 4.   Peak values of caffeine-induced Ca2+ transients. Average changes in fluorescence from soleus and EDL muscle fibers are shown. Fibers were exposed to progressively increasing concentrations of caffeine. Only the value for soleus fibers in 16 mM caffeine is statistically different from values before application of caffeine.

Identifying individual fibers in a population. Figure 5 shows the position and orientation of identifiable fibers. The images were processed to show only the fiber boundaries. Figure 5, left, shows the boundaries of the seven EDL fibers in Fig. 1. Figure 5, right, shows the boundaries of five soleus fibers in a separate experiment. Both were created by selecting pixels in one range of gray scale values and assigning them to the foreground, while assigning all of the other pixels to the background. Some of the fibers are marked by arrows to facilitate their identification in the fluorescent images in Figs. 6-9.


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Fig. 5.   Boundaries of fibers. Transmitted light images of EDL and soleus fibers were processed to show position of fiber boundaries in fluorescent images. Numbered arrows identify particular fibers.

Original images of fluorescence. Figure 6 shows original images of the EDL fibers and soleus fibers of Fig. 5, before and after the fibers were exposed to 16 mM caffeine. Forty-one consecutive frames of the EDL fibers are shown at top. Fourteen consecutive images of soleus fibers are shown at bottom. Caffeine was raised from 8 to 16 mM after frame 8 for the EDL and after frame 1 for the soleus. The images of the EDL fibers were nearly indistinguishable from one another, but the response of the soleus fibers was markedly different. Caffeine induced a large rise in Ca2+ that propagated as a wave along the length of the largest soleus fiber in the field. The velocity of the Ca2+ wave was 2.24 µm/s.


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Fig. 6.   Original fluorescent images of EDL and soleus fibers before and after exposure to 16 mM caffeine. Numeric scale ranges from 0 (complete absence) to 4096 (saturation). Top: original images of 41 consecutive frames from EDL fibers. Perfusing solution was changed from 8 to 16 mM caffeine between frames 8 and 9. Arrow at frame 1 indicates same fiber as arrows 1 and 2 in Fig. 5, left, and arrow at frame 19 of Fig. 7, top. Bottom: original images of 14 consecutive frames from soleus fibers. Perfusing solution was changed from 8 to 16 mM caffeine between frames 1 and 2.

Difference images of fluorescence. Figure 7 shows the same data as the difference between each image and its immediate predecessor. The solution perfusing the EDL fibers was changed after frame 7. Individual EDL fibers showed moderately large fluctuations in brightness, with a large variation in the time at which a fluctuation occurred. The average response of these same fibers is plotted in Fig. 2. The average of the entire population obscured the wide variation in time between addition of caffeine and a rise and fall in Ca2+ in any given fiber. Specific fibers (e.g., the EDL fiber labeled 4 in Figs. 5 and 7) responded with a delay of 88 s to 8 mM caffeine (not shown) and responded <8 s after 16 mM caffeine.


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Fig. 7.   Difference images of frames in Fig. 6. Unaltered images of EDL fibers appeared nearly identical to one another. Subtracting each one from its nearest neighbor in time produced these consecutive difference images. Regions that did not change between images are black. Caffeine (16 mM) was added between frames 7 and 8 of EDL fiber images (top) and during frame 1 of soleus fiber images (bottom). Variations in Ca2+ among individual fibers, and in same fibers at different times, can be matched with position and orientation of fiber boundaries in Fig. 5. Arrow at frame 19 indicates same fiber as arrows 1 and 2 in Fig. 5, left, and arrow at frame 1 of Fig. 6. Arrows at frame 36 indicate same fiber as arrows 3 and 4 in Fig. 5, left.

The difference images of the soleus fiber responses plotted in Fig. 3 are shown in Fig. 7, bottom. One fiber, the soleus fiber labeled by arrows 1 and 2 in Fig. 5, right, was responsible for the responses in Fig. 3. The lag between elevating caffeine to 16 mM and the response of this fiber was <8 s. In high concentrations of caffeine, the lag times for both specific EDL and soleus fibers were sometimes the same, although this could not be detected by comparing the average response of the two populations.

Fluorescence from individual fibers before caffeine. When the fibers were observed before raising the caffeine concentration, discrete, local transients of Ca2+ were distributed randomly within both EDL and soleus fibers (Figs. 8 and 9). These local transients of Ca2+ are invisible on the scale of Fig. 7, but frames 7 and 8 of the EDL fibers in Fig. 7 are also shown in Fig. 8, top and bottom, on an expanded scale. The same individual EDL fiber can now be identified in four different images (Figs. 1, 5, 7, and 8). The fiber is labeled with arrows 3 and 4 in Fig. 5, right, and with two arrows at frame 36 in Fig. 7, top. As shown in Fig. 8, bottom, this is the first fiber to brighten after elevation of caffeine. Figure 8, top and bottom, corresponds to ~800 s on the x-axis of Fig. 2, where this change is invisible. Results such as those in Figs. 7 and 8, bottom, discount the possibility that the discrete, local transients of Ca2+ might arise from some unidentified source of systemic optical noise rather than from biological events.


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Fig. 8.   Initial change in rat EDL fibers after caffeine. Difference images after expanding scale and rendering each value for Ca2+ in a third dimension are shown. Top: after 7 min in 8 mM caffeine. Bottom: <8 s after raising caffeine from 8 to 16 mM. Values are same as those in frames 7 and 8 of Fig. 7, top, immediately before and after raising caffeine concentration, respectively. White lines are guides to depth and dimension. They connect points on a grid 10 × 10 µm apart.


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Fig. 9.   Initial change in rat soleus fibers after caffeine. Difference images after expanding scale and rendering each value for Ca2+ in a third dimension are shown. Top: pattern before addition of any caffeine. Bottom: pattern 32 s after addition of 4 mM caffeine. White lines are guides to depth and dimension. They connect points on a grid 10 × 10 µm apart.

The response of soleus fibers to caffeine was strikingly different from that of EDL fibers. Figure 9, top, shows the fluorescent difference image of a soleus fiber immediately before any caffeine was applied. Figure 9, bottom, corresponds to 32 s after the first exposure to caffeine (4 mM). A very small rise corresponding to the difference between Fig. 9, top and bottom, can be seen in Fig. 3.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fundamental difference in Ca2+ release between EDL and soleus fibers. We found a fundamental difference in the way caffeine releases Ca2+ from the SR of individual, intact fast-twitch and slow-twitch rat skeletal muscle fibers. Ca2+ release was restricted to spots about one sarcomere apart before addition of caffeine (Figs. 8, top, and 9, top). The spatial dimensions of these spontaneous local Ca2+ transients were similar for both types of fiber. Each application of caffeine caused the spontaneous patterns to change. The changes differed between fast-twitch and slow-twitch fibers and also differed from the changes attributable to sarcomere motion. In EDL, release was amplified moderately but remained local when caffeine was added (Fig. 8, bottom). In soleus, however, Ca2+ release increased greatly and generated a wave that spread along the length of a fiber (Fig. 9, bottom). Ca2+ waves in slow-twitch fibers may reflect the caffeine-sensitive mechanism of CICR. Fast-twitch fibers evidently lack this mechanism for amplifying the release of Ca2+. This alone could account for the lower caffeine concentrations at which the thresholds for Ca2+ release and mechanical activity are reached in slow-twitch muscle (7).

Speed of Ca2+ release. There was a long delay between the application of low concentrations of caffeine and the onset of a significant response from a population (Figs. 2 and 3). In both EDL and soleus muscle fibers, Ca2+ can be expected to diffuse very slowly in the cytoplasm and particularly so when there are large quantities of buffering proteins or the buffering proteins are only moderately saturated (15). Parvalbumin is expressed in high concentrations in fast-twitch muscles and is nearly undetectable in slow-twitch fibers (24, 27). One might expect fast-twitch fibers to have a larger concentration of buffering proteins than slow-twitch fibers, and these may blunt the rise in Ca2+ caused by the initial application of low doses of caffeine (7). Hence, our results could also be explained if parvalbumin in EDL fibers bound Ca2+ before Ca2+ could diffuse to other ryanodine receptors and trigger CICR.

The Ca2+ waves we observed were slow compared with the speed of other signals that propagate and slow compared with the speed of events in normal excitation-contraction coupling. Ca2+ has an apparent diffusion coefficient ~55 times slower in skinned frog muscle than in water (16). We estimated a faster rate of Ca2+ movement in intact rat soleus fibers than in skinned frog fibers, from the waves, which had a velocity of ~2 µm/s or ~1 sarcomere/s. Figure 10 shows the profiles of fluorescence along the soleus fiber in Fig. 6. The natural log of the intensity is plotted as a function of distance along the axis of the fiber. The level of Ca2+ at the start of the first profile is lower than it is for all the profiles that immediately follow, which suggests that the buffering proteins were only moderately saturated before each wave. We calculated the apparent maximum diffusion coefficient from the profiles, using the equation derived by Crank (3). The diffusion coefficient in the advancing portion of the line profiles ranged from 2.7 × 10-6 to 4.1 × 10-6 cm2 · s-1, ~1.7-2.6 times slower than in water.


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Fig. 10.   Line intensity profiles of a Ca2+ wave in a soleus fiber. A line was drawn through center of soleus fiber in Fig. 6, parallel to its long axis. Natural logarithm of intensity along that line is plotted for frames 1-8 of Fig. 6, bottom, demonstrating propagation of a Ca2+ wave from left to right.

Heterogeneity of responses to caffeine. The mechanisms controlling SR Ca2+ release in different types of vertebrate skeletal muscle may be fundamentally different in several respects yet to be evaluated (e.g., Ref. 32). There may also be features of Ca2+ release from internal stores that are common to a large number of cell types. For example, the times between addition of caffeine and Ca2+ release from rat EDL fibers were very different from one another (Fig. 7). This is analogous to the Ca2+-release response of other cell types, including cells in which the variability is unlikely to be related to cell cycle differences, or the presence of multiple cell clones within a population (21).

Ca2+ channel adaptation or inactivation. The kinetics of purified skeletal muscle Ca2+-release channels in planar lipid bilayers have recently been studied by Laver and Curtis (18) with a flow method for Ca2+ that maintains a steady Ca2+ concentration for 5 s. Laver and Curtis (18) found a decline in SR Ca2+-release channel activity following activation that seems to reveal a basic channel property. Although they note that the rates of decline measured by themselves and others are too slow to be part of the regulatory mechanism in excitation-contraction coupling that involves voltage-gated Ca2+ channels, this property is fast enough to explain the adaptation/inactivation in Ca2+ release that we observed after each increment in caffeine.

Sarcomere adaptation or inactivation. Mechanical activity of rat fibers in caffeine was, in general, a short-lived phenomenon in our experiments. Our basis for speculating why caffeine-induced mechanical activity was absent comes primarily from experiments on isolated, intact frog muscle fibers. Our muscles were slack, and caffeine-induced sarcomere activity in frog muscle is strongly dependent on sarcomere length. The frequency of sarcomeric oscillations increases about threefold as a frog fiber is stretched from an average striation spacing of 2.2-2.7 µm (11). Sarcomere activity is also strongly influenced by temperature, but in the opposite sense. Below 12°C mechanical activity is independent of temperature for >1 h, but raising the temperature above 15°C abolishes oscillations in 15 min (11). The same factors might have caused mechanical activity in our unrestrained rat muscle fibers to become uncoupled from caffeine-induced Ca2+ release.

Local Ca2+ transients are not associated with sarcomere motion. The only direct imaging study of caffeine-induced sarcomere oscillation was performed on frog skeletal muscle by Kumbaraci and Nastuk (14). The modal sarcomere length of their control muscle fibers was 2.8 µm, and the modal value for fibers oscillating 1.5 h after applying caffeine was 7% shorter. Kumbaraci and Nastuk (14) found that contracting sarcomeres stretched adjoining relaxed sarcomeres and that the oscillations are generated by a limited number of myofibrillar bundles across a given fiber. Difference images of rat fibers in transmitted light showed a pattern of mechanical activity similar to that reported for frog muscle fibers (Fig. 1). The patterns of these sarcomere movements were qualitatively different from the pattern of fluorescent difference images.

The sarcomere motions we observed also had no quantitative likeness to the patterns attributed to changes in Ca2+ (Fig. 11). Sarcomere motion always produced changes that retained disjointed striated regions and striations that translated along myofibrils (Fig. 1). The transform of these transmitted light difference images clearly showed the spatial frequencies of the striations and were anisotropic in the direction of the myofibrils (Fig. 11). On the other hand, the fluorescent difference images were always free of striated or translated regions and leading or trailing edges. In addition, their power spectra were isotropic and showed no discrete frequency components, despite large changes in Ca2+ (Fig. 11).


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Fig. 11.   Frequency spectra of difference images covering period marked by horizontal line with arrowheads in Fig. 2. Images were taken 56 s before raising caffeine concentration to 16 mM (top left) and 104 s (top right) and 216 s (bottom left) after raising caffeine concentration; numbers in bottom left corner of images indicate corresponding spatial domain images in Fig. 7, top (frames 1, 20, and 36, respectively). Bottom right: spectrum of a transmitted light difference image taken at time indicated by right asterisk in Fig. 2. Corresponding spatial domain image is Fig. 1, top left.

Diffusible extracellular activator. Kumbaraci and Nastuk (14) found that a low-molecular-weight substance was released from muscle cells during the period in which sarcomere oscillations and traveling waves of mechanical activity occur in caffeine. This substance could induce propagated mechanical activity in other fibers, a finding that has since been confirmed (10-12). An extracellular diffusing messenger is evidently not a factor in rat soleus fibers that exhibited propagated Ca2+ waves, because the speed and direction of a wave was unrelated to imposed fluid flow across the fibers.

In summary, previous studies of this nature were performed on stretched, intact, whole muscle or fiber bundles, and isometric force was used to deduce underlying differences in the patterns of Ca2+ release. Although the classification of fast-twitch vs. slow-twitch fibers ignores the diversity of other measured features (24), the differences we observed are consistent with this partial division (27). Ca2+ waves in slow-twitch fibers may reflect the caffeine-sensitive mechanism of CICR. Fast-twitch fibers lack this mechanism, which may account for their lower sensitivity to caffeine.

    ACKNOWLEDGEMENTS

This research was supported by the Maimonides Research Foundation (M. K. D. Pagala), National Science Foundation Grants DMB-85-03964 and IBN-92-13160, and the Mayo Foundation (S. R. Taylor).

    FOOTNOTES

Address for reprint requests: S. R. Taylor, Mayo Foundation, 711 Guggenheim Bldg., Rochester, MN 55905-0001.

Received 27 January 1997; accepted in final form 5 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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