Effects of Caffeine at Different Temperatures on Contractile Properties of Slow-Twitch and Fast-Twitch Rat Muscles

Physiol. Res. 55: 641-652, 2006


Effects of Caffeine at Different Temperatures on Contractile
Properties of Slow-Twitch and Fast-Twitch Rat Muscles

Y. WONDMIKUN1, T. SOUKUP2, G. ASMUSSEN3

1Department of Physiology, University of Gondar, Gondar, Ethiopia, 2Institute of Physiology,
Academy of Sciences of the Czech Republic, Prague, Czech Republic and 3Carl-Ludwig-Institute of
Physiology, University of Leipzig, Leipzig, Germany

Received October 12, 2005
Accepted May 27, 2006



Summary
The slow-twitch soleus muscle (SOL) exhibits decreased twitch tension (cold depression) in response to a decreased
temperature, whereas the fast-twitch extensor digitorum longus (EDL) muscle shows enhanced twitch tension (cold
potentiation). On the other hand, the slow-twitch SOL muscle is more sensitive to twitch potentiation and contractures
evoked by caffeine than the fast-twitch EDL muscle. In order to reveal the effects of these counteracting conditions
(temperature and caffeine), we have studied the combined effects of temperature changes on the potentiation effects of
caffeine in modulating muscle contractions and contractures in both muscles. Isolated muscles, bathed in a Tyrode
solution containing 0.1-60 mM caffeine, were stimulated directly and isometric single twitches, fused tetanic
contractions and contractures were recorded at 35 °C and 20 °C. Our results showed that twitches and tetani of both
SOL and EDL were potentiated and prolonged in the presence of 0.3-10 mM caffeine. Despite the cold depression, the
extent of potentiation of the twitch tension by caffeine in the SOL muscle at 20 °C was by 10-15 % higher than that at
35 °C, while no significant difference was noted in the EDL muscle between both temperatures. Since the increase of
twitch tension was significantly higher than potentiation of tetani in both muscles, the twitch-tetanus ratio was
enhanced. Higher concentrations of caffeine induced contractures in both muscles; the contracture threshold was,
however, lower in the SOL than in the EDL muscle at both temperatures. Furthermore, the maximal tension was
achieved at lower caffeine concentrations in the SOL muscle at both 35 °C and 20 °C compared to the EDL muscle.
These effects of caffeine were rapidly and completely reversed in both muscles when the test solution was replaced by
the Tyrode solution. The results have indicated that the potentiation effect of caffeine is both time- and temperature-
dependent process that is more pronounced in the slow-twitch SOL than in the fast-twitch EDL muscles.


Key words
Rat • Slow and fast muscles • Contractile properties • Caffeine • Temperature dependence • Calcium transients


Introduction
extract of coffee beans, is being used as a constant tool in

muscle research, especially for its interaction with
Caffeine (1,3,7 trimethylxanthine), a well-known
excitation-contraction (E-C) coupling and for its ability to


PHYSIOLOGICAL RESEARCH
ISSN 0862-8408
© 2006 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Fax +420 241 062 164
E-mail: physres@biomed.cas.cz
http://www.biomed.cas.cz/physiolres

---

642 Wondmikun et al.

Vol. 55


induce complete, but reversible emptying of Ca2+ stores
slow-twitch SOL and the fast-twitch EDL muscles. Our
in the sarcoplasmic reticulum (SR). It is now evident that
results demonstrate that the effects of caffeine are
caffeine acts at the activation site of ryanodine receptors
temperature- and time-dependent processes and that they
(RyRs) in the SR Ca2+ release channel by increasing the
are more marked in the slow-twitch SOL than in the fast-
affinity for Ca2+ (for review see Hermann-Frank et al.
twitch EDL muscles.
1999, Hille 2001). Caffeine effects on the E-C coupling

process are manifested at lower concentrations (0.1-10
Methods
mM, depending on muscle type), whereas at higher

concentrations (above 10-20 mM) caffeine has a direct
The SOL and EDL muscles of 23 Wistar rats of
and reversible effect on the contractile apparatus causing
either sex (age 21-30 days, body mass 90-120 g) were
muscle contracture.
used for in vitro experiments. The young rats were used,
It was found that slow- and fast-twitch muscles
because their muscles are smaller, their diffusion
differ in their response to a lower temperature (Close and
conditions are better and their muscles survive longer in
Hoh 1968a, Buller et al. 1968, 1984, Ranatunga 1984,
vitro (Close and Hoh 1968b). The contractile properties
Ranatunga and Wylie 1989, Asmussen and Gaunitz 1989,
measured are therefore not identical, but they are quite
Barnes 1993), lyotropic (Br-, NO -3, and I-) anions comparable to those of adult rats (Close 1972). The 3- to
(Klemm et al. 1998, Gong et al. 2002, Wondmikun et al.
4-week-old rats have in the SOL muscle a slow fiber
2003) or caffeine (e.g. Weber and Herz 1969, Anwyl et
proportion of about 50-60 % and a contraction time of
al. 1984, Fryer and Neering 1989, Pagala and Taylor
about 35 ms, while 4- to 6-month-old rats have a slow
1998, Choisy et al. 2000, Hleihel et al. 2001). In muscles
fiber content of about 90 % and contraction time of about
composed mainly of slow-twitch (type I) fibers, a 40 ms. There are no significant differences in the
decrease in muscle temperature reduces the twitch tension
contraction times of the EDL between younger and older
(cold depression), whereas in muscles mainly containing
rats, because the fiber type composition does not change
fast-twitch (type IIA, IID/X or IIB) fibers, the twitch
significantly after the second month (e.g. Zacha?ová et al.
tension is enhanced (cold potentiation) (e.g. Wondmikun
2005). The experimental animals were anesthetized for
et al. 2003). The maximum tetanic force progressively
several hours with urethane (1.5 g/kg body mass i.p.) and
diminishes with decreasing temperature in all muscles
the muscles were successively excised during the
regardless of their fiber type composition (Rall and experiments.
Woledge 1990). Consequently, cooling increases the
Anesthesia with urethane lasts for up to 15 hours
twitch-tetanus ratio in the fast-twitch and decreases it in
(so called non-return narcosis). It has no influence on
the slow-twitch muscles. Therefore, investigation of the
neuromuscular transmission (Lüllmann/Mainz, personal
temperature effect can be used for classifying a given
communication). The animal is in deep anesthesia for the
muscle (Asmussen and Gaunitz 1989). Furthermore, we
whole experimental period and when measurements of
have shown that compared to normal Cl? Tyrode solution,
one muscle are terminated, the experimentator can
Br?, NO ?
3 and I? profoundly alter the contractile behavior
prepare a “fresh” muscle with an intact blood supply.
of the slow-twitch soleus (SOL) and fast-twitch extensor
After finishing the experimental analysis of the last
digitorum longus (EDL) muscles as they prolong the
muscle, the animals are sacrificed with an overdose of the
contraction and half-relaxation times, albeit to a different
anesthetic.
extent, and enhance the twitch force (Wondmikun et al.
The Ethical Principles and Guidelines for
2003). SOL and EDL muscles also differ in their Scientific Experiments on Animals were respected
response to caffeine, as the SOL muscle is more sensitive
throughout this study. The maintenance and handling of
to twitch potentiation and contractures evoked by caffeine
experimental animals followed the EU Council Directive
than the EDL muscle (Gutmann and Hanzlíková 1968,
(86/609 EEC) and the animals were treated in accordance
Isaacson et al. 1970, Singh and Dryden 1989, Adnet et al.
with principles of the Care and Use of Animals.
1993, Pagala and Taylor 1998, Lamb et al. 2001, Joumaa

and Léoty 2002).
Tyrode solution and tension recording
The aim of our study was to compare the
The isolated muscles were fixed vertically in a
combined effects of temperature and caffeine changes in
thermo-stabilized plexiglass chamber (volume 30 ml). A
modulating muscle contractions and contractures in the
continuous flow (5 ml/min) of thermo-stabilized Tyrode

---

2006

Caffeine and Contractile Muscle Properties 643




solution bubbled with a mixture of 95 % O2 and 5 % CO2
tension development of a single twitch to its peak (Pt); the
was maintained. The small volume of the chamber half-relaxation time [HRT, ms] = decay time of a single
enabled a fast exchange of the solution, e.g. during a
twitch measured from the peak (Pt) to 50 % of the tension
switch from 20 °C to 35 °C and back or between different
developed; the maximum isometric tension (T0) of a
caffeine concentrations. A complete exchange of fused tetanic contraction [mN]; stimulation frequency at
solutions lasted less than 5 s. The Tyrode solution had the
35 °C: EDL = 200 Hz, SOL = 80 Hz; at 25 °C: EDL =
following composition [mM]: NaCl 137, KCl 5, CaCl2 2,
100 Hz, SOL = 40 Hz; stimulation duration: 500 ms; the
MgCl2 1, NaH2PO4 1, NaHCO3 12, glucose 11. pH was
twitch-tetanus ratio = Pt/T0.
adjusted to 7.4, and the temperature, measured by an

electronic thermometer in the muscle chamber, was kept
Experimental protocols
constant both at 35 °C and 20 °C (maximum fluctuation
For the temperature studies, the excised muscles
of ± 0.3 °C).
were set up for recording at either 37 °C or 15 °C and
Mechanical responses were recorded isometric twitches were subsequently recorded at
isometrically using a modified mechanoelectric centigrade intervals.
transducer (51 D 17, Disa Electronic Copenhagen,
In the caffeine studies, records were obtained
Denmark) with a compliance of 0.24 mm/mN and a linear
from each muscle for about 10 min in a Tyrode solution
(± 3 %) characteristic in the range of 5-1000 mN. The
at either 35 °C or 20 °C. 35 °C served as control for
undamped resonance frequency was 950 Hz. The subsequent measurements, whereas 20 °C was chosen
amplified output signal of the transducer was displayed
because the twitch depression of slow-twitch muscles is
on an oscilloscope, recorded photographically and clearly visible and the twitch potentiation of fast-twitch
registered (basic tension) by a compensation recorder
muscles is maximal at this temperature (cf. Fig. 1). The
(response time < 0.2 s). The length of the muscle was
muscles were then superfused by a Tyrode solution
extended until twitch tension (Pt) was maximal (optimal
containing 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 5.0 or 10.0 mM
muscle length, L0).
caffeine and dynamic properties were measured after 1, 2,

3, 4, 6, 8, 10, 12 and 15 min at either temperature.
Stimulation
Thereafter, the muscles were allowed to recover from the
Neuromuscular transmission was blocked by drug effect in a normal Tyrode solution and the twitches
exposing the muscles to a Tyrode solution containing 1.5
were recorded at either 35 °C or 20 °C at the same
x 10–5 g/ml d-tubocurarine (Curarin-Asta) for 20-30 min
intervals as described above to assess the kinetics of the
before starting the experiments. The direct stimulation
recovery process at both temperatures. Then the muscle
was carried out by a transverse field of two smooth
bath was either cooled to 20 °C or warmed to 35 °C and
platinum electrodes 1 cm apart from each other, set on
the same uniformly timed procedure was repeated. At the
either side of the muscle and extending beyond its length
end of every set of twitch tension recordings, tetani were
and breadth (“massive” stimulation, Mostofsky and evoked by a 500 ms train of supramaximal stimuli at a
Sandow 1951). Because the mechanical responses depend
fusion frequency to determine the maximum tetanic
on intensity and duration of the stimulus (Close and Hoh
tension and the twitch-tetanus ratio.
1968b), a supramaximal (? 0.9 A/cm²) stimulus intensity
To study contractures, muscles were first tested
was chosen with duration of 0.1 ms.
at the lowest caffeine concentration during which records

of twitch and base line tensions were obtained (2 mM for
Parameters measured
the SOL and 5 mM for the EDL muscles; cf. Fig. 5). The
During the experiments of 2-4 h duration, the
concentration of caffeine was subsequently increased in a
amplitudes of single twitches and tetanic contractions
stepwise manner 2, 5, 10, 20, 40 and 60 mM), always
remained nearly unchanged. Only experiments in which
after a maximum contracture developed at the previous
the amplitude of a single twitch at the end of the lower concentration.
experiment was more than 90 % of that at the beginning

were analyzed. The following contractile parameters were
Statistical analysis
determined at optimal muscle length (L0): the maximum
Data are presented as means ± S.D. The paired
isometric tension (Pt) of a single twitch [mN]; the Student's t-test was used for comparison of the control
contraction time [CT, ms] = time from the onset of
and experimental data (Weber 1967).

---

644 Wondmikun et al.

Vol. 55


Results

Contractile properties of SOL and EDL at various
temperatures
The change of single twitches of SOL and EDL
muscles over a range of temperatures of 15-37 °C is
illustrated in Figure 1. Cooling of the SOL muscle
showed a monotonous gradual fall of the twitch tension
(cold depression) over the whole temperature range (Fig.
1A). Cooling of the EDL muscle was followed by a
continuous enhancement of the twitch tension (cold
potentiation) reaching a maximum at 20 °C (increase by
about 60±20 %); however, further cooling below 20 °C
decreased the extent of this maximal cold potentiation of
the twitch tension (Fig. 1A). A decrease of the
temperature of the bathing solution caused a 3 to 5 fold
prolongation of the contraction (Fig. 1B) and half-
relaxation times (Fig. 1C) in both muscles.
The contractile parameters of SOL and EDL
muscles at 35 °C and 20 °C are compared in Table 1
(N-SOL, N-EDL). Upon cooling to 20 °C, the maximum
tetanic tension of both muscles decreased by about 10 %
on the average. The decline of the tetanic tension and the
diverse sensitivity of the twitch tension upon cooling in
SOL and EDL muscles (either cold depression or
potentiation) increased the twitch-tetanus ratio in fast-
twitch EDL muscles, while it remained fairly constant in
slow-twitch SOL muscles.
The absolute temperature dependence of SOL
and EDL twitch tension was low. ƒ10 ranges between
1.02-1.17 in the SOL and 0.63-1.3 in the EDL muscle
(Table 2). ƒ

10 of the tetanic tension of both muscles was

1.07±0.02 over the whole temperature range investigated.
Fig. 1. Influence of temperature on single twitch properties of
The temperature dependence of the contraction and half-
isolated rat slow-twitch soleus (empty symbols) and fast-twitch
extensor digitorum longus muscles (filled symbols). Data are
relaxation times was greater than that of the force plotted as percentage of a control twitch at 35 °C (means ± S.D.,
parameters and both muscles showed Q10 values above 2
n = 29). Twitch tension (A), contraction time (B), half-relaxation
(Table 2). They displayed a higher temperature quotient
time (C).

at lower temperatures; the highest temperature
dependence of the contraction and especially for half-
(mathematically determined in the same manner as Q10).
relaxation time was noted for the SOL muscle between 15
Values of Q10 or f10 around 1 indicate physical processes.
and 20 °C (Table 2).
Chemical processes have values of 2 or higher (according
By convention, the temperature dependence of a
to the number and temperature dependencies of the
process is expressed as the mean change over a involved processes). The temperature coefficient (ƒ10) for
temperature range of 10 °C. According to the rule of van
twitch tension of EDL and SOL was calculated as tetanic
Hoff, the Q10 is defined for time-dependent processes,
and twitch forces (not time-dependent processes), i.e.
e.g. contraction or relaxation times. Because the they are not suitable to be expressed as Q10.
amplitude tension (twitch tension or T0) exerted by a

muscle are not time-dependent values, their temperature-
Action of caffeine at 35 °C
dependence (coefficients) are denoted as f10
A representative example (Fig. 2A) of the effect

---

2006

Caffeine and Contractile Muscle Properties 645






Fig. 2. Representative records of single twitches of isolated rat slow-twitch soleus and fast-twitch extensor digitorum longus (EDL)
muscles in standard Tyrode solution and the effect of 2 mM caffeine at 35 °C (A) and at 20 °C (B). Note the different time scale for the
EDL and soleus muscles at both temperatures.


of 2.0 mM caffeine on the time course of single twitches
more pronounced in the SOL muscles (Fig. 3C). Lower
of EDL and SOL muscles at 35 °C shows an increase of
concentrations of caffeine showed a potentiation of the
the twitch tension as well as a prolongation of contraction
twitch tension without affecting the resting tension of the
and half-relaxation times. These effects of caffeine are
preparations. At medium and higher concentrations, a
confirmed by quantitative results obtained at different
dose-dependent increase appeared in the baseline tension
caffeine concentrations (Table 1, C-SOL, C-EDL; (Fig. 5). However, a continuation of the twitch
Fig. 3A). After application of 5 mM caffeine, the twitch
potentiation throughout the contracture was observed
tension increased by about 40 % in both muscles, while
around these threshold values. At higher caffeine doses
the maximum tetanus force increased in both muscles
(>5 mM in SOL and >20 mM in EDL muscles,
only by about 10 %; the twitch/tetanus ratio thus respectively), the amplitude of the evoked twitch
increased by almost 40 % in the SOL and by about 25 %
diminished.
in the EDL muscles (Table 1). The obtained values of

twitch tensions showed that the SOL muscle was more
Action of caffeine at 20 °C
sensitive to caffeine than the EDL muscle. In the SOL
A representative example of the effect of 2.0
muscle, a significant twitch potentiation (p<0.05) was
mM caffeine (Fig. 2B), as well as quantitative evaluation
already observed at a caffeine concentration of 0.3 mM,
at different caffeine concentrations (Fig. 3B) on the time
whereas in the EDL muscle it was initiated only at higher,
course of single twitches at 20 °C shows, similarly as at
1.0 mM caffeine concentrations (Fig. 3A). Caffeine 35 °C, that the SOL muscle exhibits higher caffeine
application led to potentiation and prolongation of the
sensitivity and more pronounced increase in twitch
contraction and half-relaxation times of both muscles,
tension in comparison to the EDL muscle. Application of

---

646 Wondmikun et al.

Vol. 55




Fig. 3. Dose-response curves of the action of caffeine on single twitch contractions of isolated rat slow-twitch soleus (open symbols)
and fast-twitch extensor digitorum longus (filled symbols) muscles at temperatures 35 °C (A, C) and 20 °C (B, D). A, B: Action on
twitch tension (circles; potentiation after caffeine is expressed as percentage of control twitches recorded in normal Tyrode solution). C,
D: Action on contraction (triangles) and half-relaxation (squares) times (means ± S.D., n = 5-6).


5 mM caffeine significantly increased the twitch tension
Kinetics of caffeine potentiation and its reversibility
(more in the SOL than in the EDL muscle), even to a
Both the SOL and EDL muscles demonstrated a
greater extent than at 35 °C especially in the SOL muscle;
progressively increasing twitch tension at 35 °C when
the maximal tetanic force was also increased compared to
superfused with 2 mM caffeine. The maximum increase,
normal Tyrode solution, but the maximum was still lower
was followed by a plateau, and was achieved after
than at 35 °C (Table 1 C-SOL, C-EDL). The twitch/
6-8 min (Fig. 4A). When tested at 20 °C, both muscles
tetanus ratio thus increased in the SOL muscle at 20 oC
demonstrated a similar course of twitch potentiation, the
similarly to that at 35 °C, whereas in the EDL muscle, the
maximum twitch potentiation being attained after

twitch/tetanus ratio remained at 20 oC as in the control
12-15 min (Fig. 4B). The augmentation after application
solution without caffeine. Caffeine application also led to
and reversal of the twitch tension to the control level
a prolongation of the contraction time and especially of
upon withdrawal of caffeine thus proceeded more rapidly
the half-relaxation time (Fig. 3D, see also Fig. 2B and
at 35 °C than at 20 °C (Fig. 4A and 4B).
Table 1), similarly as at 35 °C. Although the EDL and

SOL muscles behaved at 20 °C similarly as at 35 °C, a
Caffeine contractures in SOL and EDL muscles at
higher threshold dose 0.5 mM caffeine for the SOL and 2
different temperatures
mM for the EDL was necessary to evoke a significant
In the SOL muscle, 2 mM and 5 mM caffeine
(p<0.05) potentiation, the extent of potentiation was regularly evoked a contracture at 35 °C and 20 °C,
practically comparable with the situation at 35 °C in the
respectively. The threshold concentration in the EDL
EDL. In the SOL, however, the extent of changes, muscle was considerably higher, about 20 mM of caffeine
especially after concentrations >1 mM, was much more
at both temperatures (Fig. 5). The onset of the contracture
prominent at 20 °C than at 35 °C (cf. Figs 3A and 3B).
began almost immediately after exposure of the muscles

---

2006

Caffeine and Contractile Muscle Properties 647




contracture tension (SOL: 20-40 mM, EDL: 20-60 mM),
and 4) a decrease of the contracture despite a further
increase in the caffeine dose (SOL: > 40 mM, EDL:
> 60 mM).

Discussion

Our results suggest that the potentiation effect of
caffeine is also a temperature-dependent process and that
at both temperatures it is more pronounced in the slow-
twitch SOL than in the fast-twitch EDL muscle. It is also
evident from the results that twitch tension changes in the
SOL muscles are more susceptible to caffeine and that the
contractures in the SOL muscles appear at lower
concentrations of caffeine than in the EDL muscles (cf.
also Anwyl et al. 1984, Singh and Dryden 1989, Pagala
et al. 1994, Choisy et al. 2000, Lamb et al. 2001). On the
other hand, caffeine produced only a relatively small
increase (10-14 %) in maximum tetanic tension,
irrespective of the muscle type and temperature. These
data are in good agreement with the suggestion of Fryer
and Neering (1989) who stated that it is likely that the
myofilaments are saturated with Ca2+ during the tetanus
and that a further increase in intracellular Ca2+ level
would produce no further increase in force.
The observed effects of caffeine were

Fig. 4. Time courses of the maximal twitch tension of soleus
completely reversible. Sorenson et al. (1986)
(open circles) and extensor digitorum longus (filled circles)
demonstrated that the Ca2+-release from the sarcoplasmic
muscles after application (?) and washing (?) of 2 mM caffeine
reticulum and the inhibition of Ca2+-uptake induced by
at 35 °C (A; n = 6) or at 20 °C (B; n = 5). The potentiation is
expressed as percentage of control twitches recorded in normal
caffeine are rapidly reversible in skinned psoas and EDL
Tyrode solution (means ± S.D.).
muscle fibers. Nevertheless, there is a distinct


dependence of the reversibility on temperature, as we
to the appropriate drug concentration, but the time to
have shown that cooling slowed down the speed of
maximum contracture varied depending on the caffeine
muscle recovery after caffeine treatment apparently due
concentration.
to a slower reuptake of Ca2+ into the SR by the Ca2+-
In the SOL muscles, the maximum contracture
pump.
tension at 35 °C and 20 °C was already obtained at 20
A further difference between the SOL and EDL
and 40 mM caffeine, respectively, whereas EDL muscles
muscles emerged when we compared extent of the twitch
required 40 mM and 60 mM caffeine for the same effect
tension potentiation produced by caffeine at 35 °C and
(Fig. 5). After reaching their maximum, the contractures
20 °C (cf. Table 1). The interaction of these two muscle
declined in both muscles despite a further rise in the
tension modulating factors showed that despite the cold
caffeine concentration. In general, both muscles depression by about 10 %, the extent of potentiation of
demonstrated four distinct phases of reaction to increased
the twitch tension by caffeine in the SOL muscle was
caffeine concentrations (cf. Fig. 5): 1) Twitch tension
higher at 20 0C than at 35 °C (cf. Table 1 N-SOL and C-
potentiation with no detectable change in the resting
SOL). On the other hand, in the EDL muscle, caffeine
tension (SOL: 2-5 mM, EDL: 5-10 mM), 2) caffeine
further increased cold potentiation of both temperatures
contracture appearing parallel to a further potentiation of
(Table 1 N-EDL, C-EDL). Furthermore, the difference in
twitch tension (SOL 5-10 mM, EDL: 10-20 mM), 3) a
the extent of twitch potentiation of the SOL muscle
decline of the twitch tension with a further rise in between 35 °C and 20 °C increased with increasing

---

648 Wondmikun et al.

Vol. 55




Fig. 5. Typical changes in baseline tension and the twitch amplitude of a rat extensor digitorum longus muscle exposed to different
caffeine concentrations (5-60 mM) at 35 °C. Exchanges of normal Tyrode solution to solutions with stepwise increasing caffeine
concentration are indicated by arrows.


concentration of caffeine and was thus most evident at
calsequestrin, exclusively when phosphorylated, led to a
5 and 10 mM caffeine concentrations (cf. Figs 3A and
fivefold increase of open probability of the RyRs and
3B). This may indicate that the increased intracellular
caused a twofold increase of the RyRs open time
Ca2+-concentration by caffeine would outweigh the cold
(Szegedi et al. 1999). As calsequestrin exists in fast and
depression in the slow SOL muscle, probably by slow/cardiac isoforms, similarly as some other Ca2+
providing additional Ca2+ for myosin phosphorylation and
binding proteins (for review see Berchtold et al. 2000), it
transition of cross-bridges into the force-generating state
is suggestive that different calsequestrin isoforms can
(Metzger et al. 1989, Sweeney and Stull 1990).
partly contribute to the observed differences of caffeine
Caffeine opens Ca2+-channels or increases the
effect in slow- and fast-twitch muscles.
probability of opening the channels via the RyRs. At low
At higher doses, the liberation of Ca2+ from the
doses this effect is small and leads to twitch potentiation
SR is so high that it couples directly to the contractile
by normal depolarization or by direct stimulation, but not
apparatus and evokes tension without any electrical
to a contracture. A subcontracture dose of caffeine stimulation (contractures). The resting tension of a
potentiates the twitches of both muscles in a muscle, however, is enhanced with an increasing dose of
concentration-dependent manner and the first measurable
caffeine. This progression of the twitch potentiation
twitch potentiation was observed at 0.3 and 1.0 mM
accompanied with contractures elicited by intermediate
caffeine for SOL and EDL muscles, respectively, which
caffeine doses (cf. also Connett et al. 1983) suggests that
appears to be consistent with earlier reports (Fryer and
potentiation and contractures are probably caused by
Neering 1989, Choisy et al. 2000, Lamb et al. 2001).
related mechanism(s) of the drug effect on sarcoplasmic
Using isolated skinned muscle fibers, these authors Ca2+-transport. To verify this, skinned fiber preparations
demonstrated dose-dependent enhancement of the force
in which Ca2+-transients would be simultaneously
and slowing of relaxation. Furthermore, they argued that
recorded at various temperatures should be used.
these effects are the result of an increased Ca2+-release
The present results have confirmed that the
and decreased Ca2+-uptake of the SR as well as temperature sensitivity of the rat EDL and SOL muscles
diminished myoplasmatic Ca2+-buffering or a markedly differs (Close and Hoh 1968a, Buller et al.
combination of these factors. It was suggested that 1968, 1984, Ranatunga 1984, Ranatunga and Wylie 1989,
caffeine induces a release of Ca2+ from calsequestrin,
Asmussen and Gaunitz 1989, Barnes 1993). The
which is the main Ca2+-binding protein of the SR, with a
enhancement of single twitch tension is a typical response
high capacity and a low affinity for Ca2+ (Scott et al.
of the rat EDL muscle upon cooling to 20 °C. On the
1988, Ikemoto et al. 1991). From these experiments it
contrary, cooling decreases the twitch tension of the rat
was concluded that the Ca2+-dependent conformational
SOL muscle by about 10 %. A cold depression of the
change of calsequestrin causes a change in the shape of
SOL muscle of various mammals was reported, whereby
SR membrane proteins, including the RyRs, and that
the depression was directly proportional to the fiber

---

2006

Caffeine and Contractile Muscle Properties 649




Table 1. Temperature dependence of contractile properties of rat slow-twitch soleus (N-SOL) and fast-twitch extensor digitorum longus
(N-EDL) muscles and the additional influence of 5 mM caffeine (C-SOL and C-EDL)


N-SOL (n = 30)
C-SOL (n =6)
N-EDL (n = 29)
C-EDL (n= 6)
Parameters
35 °C
20 °C
35 °C
20 °C
35 °C
20 °C
35 °C
20 °C


CT [ms]
29.0±2.7 100.2±5.6
57.9±4.9 175.6± 4.7 12.5±1.5 52.9±0.1 18.7±2.3 59.1±3.8
HRT [ms] 32.3±2.8
138.0±20.1
81.9±5.1

245.4±12.8 13.2±1.6 59.3±11.2 21.1±2.9 102.3±4.6
P
1
t/Pt35 1 0.89±0.03
1.46±0.04
1.51±0.05
1
1.61±0.18 1.39±3.8 1.99±6.9
T
2
0/T035
1 0.90±0.04
1.11±0.04
1.08±0.05
1
0.87±0.04
1.10±0.03
1.04±0.4
Twitch/tetanus 0.13±0.03 0.14±0.02
0.19±0.04 0.20±0.04
0.15±0.06 0.29±0.07
0.19±0.04 0.29±0.07


Data are means ± S.D. CT = contraction time, HRT = half-relaxation time, Pt = twitch tension, 1expressed as a fraction of the twitch
tension developed at 35 °C in normal Tyrode, T0 = tetanic tension, 2expressed as a fraction of the tetanic tension developed at 35 °C in
normal Tyrode solution; significant difference (P<0.01) between 20 °C and 35 °C of homologous muscles were found in all cases.


Table 2. The temperature coefficient (ƒ10) of muscle twitch force and the temperature quotient (Q10) of time parameters at different
temperature ranges.


15-20 °C
21-30 °C
31- 37 °C
Temperature dependency ratio EDL
SOL
EDL
SOL
EDL
SOL


ƒ10 [Pt] 1.30±0.11
1.17±0.12
0.71±0.13
1.10±0.10 0.63±0.13
1.02±0.10
Q10 [CT] 3.12±0.17
3.25±0.22
2.73±0.15
3.10±0.17 2.11±0.15
2.09±0.16
Q10 [HRT] 3.64±0.26
5.80±0.32
3.12±0.24
4.03±0.26 2.81±0.20
2.01±0.20


Data are means ± S.D., n = 29. EDL = extensor digitorum longus muscle, SOL = soleus muscle, Pt = twitch amplitude, CT = contraction
time, HRT = half-relaxation time


composition of muscles, i.e. to the percentage of slow and
The temperature dependence of various
fast twitch fibers (Asmussen and Gaunitz 1989, mechanical parameters of muscle contraction is very
Wondmikun et al. 2003; for fiber type composition, see
different. The observed Q10 and ƒ10 values range from
e.g. Soukup et al. 1979, 2002, Vadászová et al. 2004,
nearly 0.5 to 5.8 (from a negative up to very strong
2006, Zacha?ová et al. 2005). Cold depression is more
positive temperature dependence). The positive and
pronounced in SOL muscles of cats and guinea pigs
negative temperature effects on muscle force are,
composed almost exclusively of slow-twitch fibers however, small, ƒ10 lying close to 1.0 (0.6-1.3; Table 2,
(Henneman et al. 1968, Asmussen and Gaunitz 1989).
cf. Bennett, 1984). The Q10 values for the contraction and
We have confirmed that the tension developed during
half-relaxation times found in this study, were above 2,
unfused or fused tetanic contractions is also temperature
indicating a moderate to high temperature sensitivity (cf.
sensitive. The tension output of fused tetani of both
Rall and Woledge 1990). There is evident correlation
muscles tested was about 10 % higher at 35 °C than at 20
between temperature dependent isometric twitch
°C. Investigation of tetanic contractions over a wide
potentiation and a myosin phosphorylable light chain
range of temperatures have shown that muscle tension is
content (Mannig and Stull 1982, Palmar and Moore 1989,
nearly constant between 30-37 °C and decreases Moore et al. 1990), however, the basis of the different
progressively below 30 °C (Bennett 1984, Kössler and
temperature sensitivity between fast-twitch and slow-
Küchler 1987, Asmussen and Gaunitz 1989). twitch fibers are not fully understood. Apart from the
Temperatures between 30-37 °C are physiological in phosphorylation mechanism which would possibly
mammalian muscles and the constancy of their force
explain the change in muscle force parameters, there may
output in this range is of functional significance because
exist some other biochemical factors such as the myosin-
motor units mainly use tetanic contractions in ATPase, the Ca2+-ATPase of the SR (SERCA) with
physiological movements in vivo.
different temperature-sensitivities operating in parallel

---

650 Wondmikun et al.

Vol. 55


and promoting muscle contraction and relaxation Acknowledgements
processes. The reaction rates of these factors are subject
Authors would like to thank R. Rätze and S. Bähnisch for
to temperature regulation, as biochemical reactions are
excellent technical help and Dr. P. Hník for critical
slowed by reduced temperature with a Q10 > 2.0 (Prosser
reading and valuable comments during preparation of the
1973). This reduction affects the rate at which actin and
manuscript. This study was supported by the Deutsche
myosin filaments can slide past one another and this
Forschungsgemeinschaft, Schwerpunkt Muskelforschung
presumably accounts for changes in contraction and a
(As 74/1-2), MYORES No. 511978 and GA?R
half of relaxation times (Barnes 1993).
304/05/0327 grants and by the Research project AV0Z

50110509.

References

ADNET PJ, BROMBERG NL, HAUDECOEUR G, KRIVOSIC I, ADAMANTIDIS MM, REYFORD H, BELLO N,
HARBER RMK: Fiber-type caffeine sensitivities in skinned muscle fibers from humans susceptible to
malignant hyperthermia. Anesthesiology 78: 168-177, 1993.
ANWYL R, BRUTON JD, MCLOUGHLIN JV: The effect of external potassium, multivalent cations and temperature
on caffeine contractures in rat skeletal muscle. Br J Pharmacol 82: 606-614, 1984.
ASMUSSEN G, GAUNITZ U: Temperature effects on isometric contractions of slow and fast twitch muscles of
various rodents – dependence on fibre type composition: a comparative study. Biomed Biochim Acta 48: S536-
S541, 1989.
BARNES WS: Extracellular calcium and the inotropic effect of cooling on isolated skeletal muscle. J Therm Biol 18:
53-59, 1993.
BENNETT AF: Temperature dependence of muscle function. Am J Physiol 247: R217-R229, 1984.
BERCHTOLD MW, BRINKMEIER H, MÜNTENER M: Calcium ion in skeletal muscle: its crucial role for muscle
function, plasticity, and disease. Physiol Rev 80: 1215-1265, 2000.
BULLER AJ, RANATUNGA KW, SMITH JM: The influence of temperature on the contractile characteristics of
mammalian fast and slow skeletal muscles. J Physiol Lond 196: 82, 1968.
BULLER AJ, KEAN CJ, RANATUNGA KW, SMITH JM: Temperature dependence of isometric contractions of cat
fast and slow skeletal muscles. J Physiol Lond 355: 21-31, 1984.
CHOISY S, HUCHET-CADIOU C, LÉOTY C: Differential effects of 4-chloro-m-cresol and caffeine on skinned fibers
from rat fast and slow skeletal muscles. J Pharmacol Exp Ther 294: 884-893, 2000.
CLOSE RI: Dynamic properties of mammalian skeletal muscle. Physiol Rev 52: 129-197, 1972.
CLOSE RI, HOH JFY: Influence of temperature on isometric contractions of rat skeletal muscles. Nature 217: 1179-
1180, 1968a.
CLOSE RI, HOH, JFY: The after-effects of repetitive stimulation on the isometric twitch contraction of rat fast skeletal
muscle. J. Physiol Lond 197: 461-478, 1968b.
CONNETT RJ, UGOL LM, HAMMACK MJ, HAYS ET: Twitch potentiation and caffeine contracture in isolated rat
soleus muscle. Comp Biochem Physiol C 74: 349-354, 1983.
FRYER MW, NEERING IR: Action of caffeine on fast- and slow-twitch muscles of the rat. J Physiol Lond 416: 435-
454, 1989.
GONG X, BURBRIDGE SM, COWLEY EA, LINDSELL P: Molecular determinants of Au(CN) -2 binding and
permeability within cystic fibrosis transmembrane conductance regulator Cl- channel pore. J Physiol Lond 540:
39-47, 2002.
GUTMANN E, HANZLÍKOVÁ V: Contracture response of fast and slow mammalian muscles. Physiol Bohemoslov
15: 404-414, 1968.
HENNEMAN E, MACPHEDRAN AM, WUERKER RB: Properties of motor units in a homogenous red muscle
(soleus) of the cat. J Physiol Lond 28: 71-84, 1968.
HILLE B: Ion Channels of Excitable Membranes. Sinauer Associates, Inc, Sunderland, MA, USA, 2001, pp 814.

---