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
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ABSTRACT |
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
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INTRODUCTION |
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+.
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MATERIALS AND METHODS |
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).
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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 |
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.
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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).

View larger version (211K):
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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.
 |
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