Imaging caffeine-induced Ca21 transients in individual
fast-twitch and slow-twitch rat skeletal muscle fibers
MURALI K. D. PAGALA1 AND STUART R. TAYLOR2
Research Laboratory, Maimonides Medical Center,
Brooklyn, New York 11219-2999; and 2Department of Pharmacology,
Mayo Foundation, Rochester, Minnesota 55905-0001
1Neuromuscular
dissimilar caffeine sensitivity; calcium release from sarcoplasmic reticulum; parvalbumin
widely to bypass voltage-gated
excitation-contraction coupling in skeletal muscle, directly gate Ca21-release channels in the sarcoplasmic
reticulum (SR), and generate Ca21-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 Ca21
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 Ca21-activated force in stretched
skinned fibers, and large doses of caffeine depress
maximum Ca21-activated force and depress the activity
of several enzymatic reactions (26, 38). Caffeine potentiates the mechanism of Ca21-induced Ca21 release
(CICR), if the Ca21 concentration in the SR is measured
by the isometric force resulting from the application of
CAFFEINE HAS BEEN USED
a high concentration of caffeine (5). The total Ca21
content of the SR in skinned fibers has also been
measured by equilibrating fibers with Ca21 buffers,
followed by lysis. This technique shows that the SR of
fast-twitch fibers is only one-third full at the myoplasmic Ca21 concentration of a resting fiber, whereas the
SR of slow-twitch fibers is saturated with Ca21 (8).
However, results using skinned fiber tension development to monitor Ca21 release have not uniformly
confirmed the assumption that force reflects Ca21 release. Some investigators have concluded that Ca21
release from slow-twitch fiber SR is more sensitive to
caffeine than Ca21 release from fast-twitch fiber SR,
whereas others have concluded that the Ca21 sensitivity of the myofilaments is the critical factor, rather than
Ca21 release (30, 36).
Many studies of Ca21-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 Ca21-release channels in
lipid bilayers show that the channels are activated by
Ca21 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
Ca21 activation of ryanodine receptors is followed by
their adaptation or inactivation at constant Ca21 concentration. Ryanodine receptors may be regulated by Ca21dependent activation and Ca21-dependent inhibition
mechanisms that gate independently (18).
The first systematic study of caffeine effects on Ca21
in wholly intact mammalian skeletal muscle measured
changes in Ca21 from average responses of populations
of fibers. The results led to the suggestions that fasttwitch SR Ca21-release channels are less sensitive to
caffeine than those of slow-twitch fibers and that
parvalbumin in fast-twitch muscles may buffer Ca21
released by low concentrations of caffeine and prevent
any useful rise in Ca21 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
0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society
C623
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Pagala, Murali K. D., and Stuart R. Taylor. Imaging
caffeine-induced Ca21 transients in individual fast-twitch
and slow-twitch rat skeletal muscle fibers. Am. J. Physiol.
274 (Cell Physiol. 43): C623–C632, 1998.—Fast-twitch and
slow-twitch rat skeletal muscles produce dissimilar contractures with caffeine. We used digital imaging microscopy to
monitor Ca21 (with fluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study
this difference. Changes in Ca21 in individual fibers were
markedly different from average responses of a population.
All fibers showed discrete, nonpropagated, local Ca21 transients occurring randomly in spots about one sarcomere
apart. Caffeine increased local Ca21 transients and sarcomere
motion initially at 4 mM in soleus and 8 mM in extensor
digitorum longus (EDL; ,23°C). Ca21 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 Ca21 transients. In soleus fibers,
16 mM caffeine greatly increased Ca21 release and produced
propagated waves of Ca21 (,1.5–2.5 µm/s). Ca21 waves in
slow-twitch fibers reflect the caffeine-sensitive mechanism of
Ca21-induced Ca21 release. Fast-twitch fibers possibly lack
this mechanism, which could account for their lower sensitivity to caffeine.
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MUSCLE SENSITIVITY TO CAFFEINE
and a population, we studied wholly intact rat muscle
fibers, with the milieu of the SR Ca21 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 Ca21 transients. Our results show that Ca21
transients from a population of cells obscure the large
variation in local Ca21 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
Ca21.
Rat muscles and fiber dissection. The data were obtained
from five soleus and five extensor digitorum longus (EDL)
muscles isolated from five anesthetized male SpragueDawley 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-N8-2-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 5 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.
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MATERIALS AND METHODS
Imaging Ca21. The microscope was a Zeiss IM-35 fitted
with an all-quartz pathway and a Nikon quartz, ultraviolettransmitting objective (320, 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 3 113-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 220°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 Ca21 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 Ca21 ) 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-tonoise 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 Ca21
MUSCLE SENSITIVITY TO CAFFEINE
indicator dye fluorescence changes occur over one plane of an
image obtained by confocal microscopy (17, 35).
RESULTS
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 Ca21
concentration (37). Apparently, the mechanical sensitivity to low concentrations of Ca21 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
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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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 6 0.02 µm (n 5 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).
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
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MUSCLE SENSITIVITY TO CAFFEINE
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).
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 Ca21 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 Ca21 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).
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 ,
Fig. 3. Effect of caffeine on Ca21 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.
P , 0.05). Ca21 always fell to the control value or less
before the next highest dose of caffeine was introduced.
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.
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
Fig. 4. Peak values of caffeine-induced Ca21 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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Fig. 2. Effect of caffeine on Ca21 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.
MUSCLE SENSITIVITY TO CAFFEINE
C627
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.
ent. Caffeine induced a large rise in Ca21 that propagated
as a wave along the length of the largest soleus fiber in
the field. The velocity of the Ca21 wave was 2.24 µm/s.
Difference images of fluorescence. Figure 7 shows the
same data as the difference between each image and its
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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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 differ-
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MUSCLE SENSITIVITY TO CAFFEINE
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 Ca21 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.
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 Ca21
were distributed randomly within both EDL and soleus
fibers (Figs. 8 and 9). These local transients of Ca21 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 Ca21 might arise from
some unidentified source of systemic optical noise
rather than from biological events.
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.
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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 Ca21 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.
MUSCLE SENSITIVITY TO CAFFEINE
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mere motion. In EDL, release was amplified moderately but remained local when caffeine was added (Fig.
8, bottom). In soleus, however, Ca21 release increased
greatly and generated a wave that spread along the
length of a fiber (Fig. 9, bottom). Ca21 waves in slowtwitch fibers may reflect the caffeine-sensitive mechanism of CICR. Fast-twitch fibers evidently lack this
mechanism for amplifying the release of Ca21. This
alone could account for the lower caffeine concentrations at which the thresholds for Ca21 release and mechanical activity are reached in slow-twitch muscle (7).
Speed of Ca21 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
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Fig. 8. Initial change in rat EDL fibers after caffeine. Difference
images after expanding scale and rendering each value for Ca21 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 3 10 µm
apart.
DISCUSSION
Fundamental difference in Ca21 release between EDL
and soleus fibers. We found a fundamental difference in
the way caffeine releases Ca21 from the SR of individual, intact fast-twitch and slow-twitch rat skeletal
muscle fibers. Ca21 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 Ca21 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 sarco-
Fig. 9. Initial change in rat soleus fibers after caffeine. Difference
images after expanding scale and rendering each value for Ca21 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 3 10 µm apart.
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MUSCLE SENSITIVITY TO CAFFEINE
Fig. 10. Line intensity profiles of a Ca21 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 Ca21
wave from left to right.
caffeine and Ca21 release from rat EDL fibers were very
different from one another (Fig. 7). This is analogous to
the Ca21-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).
Ca21 channel adaptation or inactivation. The kinetics of purified skeletal muscle Ca21-release channels in
planar lipid bilayers have recently been studied by
Laver and Curtis (18) with a flow method for Ca21 that
maintains a steady Ca21 concentration for 5 s. Laver
and Curtis (18) found a decline in SR Ca21-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 Ca21 channels, this property is fast
enough to explain the adaptation/inactivation in Ca21
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 Ca21 release.
Local Ca21 transients are not associated with sarcomere motion. The only direct imaging study of caffeineinduced 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 Ca21 (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
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fibers, Ca21 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 Ca21
caused by the initial application of low doses of caffeine
(7). Hence, our results could also be explained if parvalbumin in EDL fibers bound Ca21 before Ca21 could
diffuse to other ryanodine receptors and trigger CICR.
The Ca21 waves we observed were slow compared
with the speed of other signals that propagate and slow
compared with the speed of events in normal excitationcontraction coupling. Ca21 has an apparent diffusion
coefficient ,55 times slower in skinned frog muscle
than in water (16). We estimated a faster rate of Ca21
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 Ca21 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 3 1026 to 4.1 3 1026 cm2 · s21, ,1.7–2.6 times slower
than in water.
Heterogeneity of responses to caffeine. The mechanisms controlling SR Ca21 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 Ca21 release from internal stores that are common to a large number of cell
types. For example, the times between addition of
MUSCLE SENSITIVITY TO CAFFEINE
Ca21 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 Ca21 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). Ca21 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.
This research was supported by the Maimonides Research Foundation (M. K. D. Pagala), National Science Foundation Grants DMB-8503964 and IBN-92-13160, and the Mayo Foundation (S. R. Taylor).
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.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
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 Ca21 (Fig. 11).
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
C631
C632
MUSCLE SENSITIVITY TO CAFFEINE
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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