Enhancement of twitch force by stretch in a nerve-skeletal muscle preparation of the frog Rana porosa brevipoda and the effects of temperature on it
Department of Biology, Faculty of Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan
* Author for correspondence (e-mail: tsuchiya{at}kobe-u.ac.jp)
Accepted 23 August 2004
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Summary |
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Key words: stretch, force enhancement, nerve-muscle preparation, synaptic transmission, thermal acclimation, nerve stimulation, frog, Rana brevipoda
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Introduction |
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The propagation mechanism of excitation in the nerve-muscle junction is
known to be chemical transmission, where transmitter substances at synapses
are released from the synaptic vesicles accumulated within the nerve endings
(Katz, 1996), and the effects
of various factors on nerve propagation have been reported; for example, the
dependence of transmission efficiency on temperature
(Adams, 1989
;
Balnave and Gage, 1974
;
Barrett et al., 1978
;
Barrett and Stevens, 1972
;
Barton and Cohen, 1982
;
Katz and Miledi, 1965
;
Molgo and Van der Kloot, 1991
;
Van der Kloot and Cohen, 1984
)
and on muscle length (Chen and Grinnell,
1995
,
1997
;
Fatt and Katz, 1952
;
Hutter and Trautwein, 1956
;
Ruff, 1996
,
2003
;
Turkanis, 1973
;
Ypey et al., 1974
;
Ypey and Anderson, 1977
). Most
experiments, however, have been performed under conditions where muscle
movement was inhibited by a blocker such as curare or by specific ionic
conditions, and the mechanical phenomenon has only been mentioned briefly in a
few papers (Chen and Grinnell,
1997
; Hutter and Trautwein,
1956
).
In the present study, we intended to explore new aspects of force enhancement by stretch using nerve-skeletal muscle preparations isolated from frogs, firstly because the phenomenon seemed to be closely related to physiological functions in situ and, further, because we discovered in early stages of the experiment that the phenomenon was remarkably affected by the temperature at which the frogs were kept, i.e. the thermal acclimation.
In the present study we show that the stretch-induced force enhancement in a nerve-muscle preparation is caused by the increased transmission rate between nerve and muscle and that this phenomenon is strongly affected by the temperature at which the frogs were kept.
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Materials and methods |
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Stimulation
Electronic stimulation to contract the muscle was applied either on the
nerve (indirect stimulation) or on the muscle (direct stimulation). Indirect
stimulation was carried out using a pair of platinum circular wires (separated
by 3 mm) around a nerve bundle. The surface of the nerve between the two
electrode wires was filled with the mixture of vaseline and oil for effective
stimulation. For direct stimulation a pair of platinum plate electrodes was
placed either side of a muscle.
A single pulse or a train of pulses generated by an electronic stimulator (SEN-3301, SEN-7203, Nihon Kohden, Tokyo, Japan) was delivered to induce a twitch or a fused tetanus of 1 s duration. For direct stimulation, the Ringer solution contained curare (d-tubocurarine chloride pentahydrate, 10-4 mol l-1). Rectangular pulses (0.03-1.0 ms in duration) of sufficient voltage, twice as strong as the threshold level, were applied through an isolator (SS-104J, SS-202J, Nihon Kohden, Tokyo, Japan) for both direct and indirect stimulation. The pulse frequency for tetanus was changed appropriately according to the temperature (20-25 Hz, 35-45 Hz and 50-80 Hz at 4±0.5, 12±0.5 and 22±0.5°C, respectively).
Recording of signals and stretch of muscle
The force was measured either by a force transducer (resonant frequency, 1
kHz) or by the feedback signal from a servomotor system (DPS-265, Diamedical
System, Tokyo, Japan) (Fig.
1B). The servomotor was also used to apply step increases in
length from the resting length (l0) and a stretch was
applied in between twitches. The time for a length step was usually 5 ms, but
was changed appropriately when a slow-ramp stretch was necessary. On
application of a stretch, the twitch force amplitudes were measured after the
passive resting force had become constant. The force per cross-sectional area
was estimated by calculating the area from the long and short diameters of a
whole muscle, assuming that the cross-section of iliofibularis muscle was
elliptical in shape. Forces developed by indirect stimulation were expressed
as values relative to the forces obtained by direct stimulation.
The signals from force and displacement transducers were displayed simultaneously on digital oscilloscopes (Model 310, Nicolet, Madison, WI, USA; DL716, Yokogawa, Tokyo, Japan) and a pen recorder (RTA-1100M, Nihon Kohden, Tokyo, Japan). To record the extracellular electrical signals from sciatic nerve or muscle, a suction electrode made of polyethylene tube (Suction electrode, A-M Systems, Carlsborg, WA, USA) was used, sucked onto the region near the tendon on the muscle surface for less movement, or on the surface of the nerve bundle, and another electrode was placed in the bath. The electrical signals were amplified by an extracellular amplifier (DPA-1000, Diamedical, Tokyo, Japan) and displayed on a digital oscilloscope.
Statistics
Values in the text and the figures are given as the mean ± standard
deviation (S.D.). Statistical differences between the values were
determined by either Student's t-test or one-way analysis of variance
(ANOVA) followed by the Scheffé post hoc test.
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Results |
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We compared the amplitude of force produced by indirect stimulation with that obtained by direct stimulation (Figs 2 and 3). The twitch force obtained by indirect stimulation was approximately one tenth of that from direct stimulation at 4°C, them were significant (P<0.01). By contrast, tetanic forces obtained by direct and indirect stimulation at each temperature were not different, though the rate of force development was slower on indirect stimulation at the lower temperature (Fig. 2). Thus the intensities of direct and indirect stimulations were sufficiently strong to activate all nerve and muscle fibres.
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Enhancement of twitch force by stretch
Fig. 4 illustrates the
changes in twitch force on application of a stretch. The stretch was applied
in between twitches, that is, to silent muscle, as shown in
Fig. 7A. On indirect
stimulation, higher twitch forces than those obtained before a stretch
superimposed on the resting force were observed after a stretch, and they
returned immediately to base level after the end of a stretch. On direct
stimulation, however, the forces did not change appreciably before and after a
stretch.
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We examined the relationship between muscle length and force production in directly and indirectly stimulated muscles (Figs 5 and 6). On direct stimulation, the twitch forces decreased at all temperatures at longer lengths than at the resting length (l0) (Fig. 5A) and were similar to those seen in tetanus (Fig. 5B). By contrast, on indirect stimulation at 4°C (Fig. 6), the enhanced forces increased with the increase in a length step and approached the regression line of the length-force relationship at approx. 1.2 x l0. At 12°C, the enhanced forces were higher at l x l0 and approached the regression line at around 1.10-1.15 x l0. At 22°C, very high relative forces (0.6-0.8) were developed even at l0 and approached the regression line at 1.05 x l0, after which the force decreased along the regression curve at longer lengths. Thus enhanced forces increased with the stretch amplitudes but followed the length-force relationship and decreased at long lengths.
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Effects of velocity of stretch on twitch force
It is known that the residual force after stretch in tetanus depends not on
the velocity but on the stretch amplitude (Edman et al.,
1978,
1982
). In the present
experiment, the effects of stretch velocity on twitch force enhancement were
examined (Fig. 7). Two
stretches at different velocities (fast and slow) from the resting length
(l0) were applied to a muscle between twitches
(Fig. 7A). The amplitude of
enhanced twitch forces following the two different stretches is almost same
(Fig. 7B).
Fig. 7C shows that the form of
the force development is the same for both stretches, which suggests that the
enhancement of twitch force is independent of stretch velocity.
Effects of the maintenance temperature of frogs on force enhancement
The results discussed so far were obtained from frogs kept at room
temperature. We attempted to keep frogs at a low temperature (4°C) for
more than 2 months and the experiments were conducted at this low temperature.
The responses shown in Fig. 8
obtained from the frogs kept at 4°C were very similar to those shown in
Fig. 6 at the experimental
temperature of 22°C from frogs kept at room temperature.
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Twitch forces at the resting length were compared after different periods of maintenance at 4°C (Fig. 9). It is clearly demonstrated that the twitch forces were higher when frogs had been kept for longer periods at 4°C. The force obtained from frogs kept at 4°C for more than 2 months was significantly higher than that from frogs kept at room temperature (P<0.01).
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Electrical signals from nerve and muscle
The results so far described seem to be explained either by a change in
conduction at the neuromuscular junction or by a change in the excitability of
nerve itself. We therefore measured the electrical signals from nerve and
muscle (Fig. 10). In muscle
(Fig. 10B), significantly
higher action potentials (15.00±3.29 mV) were observed after a stretch
than before (4.83±0.71 mV) (P<0.01), whereas the heights of
the action potentials observed in nerves before and after a stretch at 4°C
were the same (Fig. 10A).
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Discussion |
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A quick stretch enhanced twitch force when muscle was stimulated
indirectly, whereas the enhancement was not observed in directly stimulated
muscle (Fig. 4). Therefore,
stretch was thought to cause the force enhancement by changing the conduction
between nerve and muscle. The preparations used in the present experiment were
whole muscles with nerves, and stretch might result in an increase in the
number of activated muscle fibres. This idea is also supported by the
electrical activities recorded from muscle, which were augmented by a stretch,
whereas action potentials recorded from nerve were the same before and after a
stretch (Fig. 10). Some
investigators have shown the augmentation of EPP and mEPP by stretch. Chen and
Grinnell (1995,
1997
) put forward the
hypothesis that augmentation was induced by tension on integrins in the
presynaptic membrane that was transduced mechanically into changes in the
position or conformation of one or more molecules involved in neurotransmitter
release. This hypothesis could also explain the enhancement of twitch force
observed in the present study.
Functional importance of the phenomena in situ
In the present study, muscles were stretched from l0
(the resting length) to 1.2 x l0. The physiological
range within which muscle works in situ is thought to be at least
between 0.95 and 1.15 x l0, and the variations in
length that may occur in the body lie within the range of the present length
change. The efficiency of neuromuscular transmission could therefore vary
during the course of voluntary muscular movement and this could be of
functional significance when fibres exist on the subliminal fringe of
excitation. When a muscle is cooled, some muscle fibres may be just below the
threshold and a stretch could evoke contraction by facilitating the
transmission. In poikilothermic animals such as frogs, force enhancement by
stretch may be of basic physiological importance when the temperature
decreases rapidly as, if the low temperature continues, frogs adapt to the
environment by thermal acclimation and can move actively. The physiological
significance of this effect has already been noted
(Hutter and Trautwein,
1956).
In homeothermic animals, by contrast, the efficiency of conduction in
neuromuscular junction is kept constant (Ruff,
1996,
2003
). The nature of the
transmission is expressed by the safety factor (SF);
SF=EPP/(EAP-RP), where EPP is the endplate potential
amplitude, RP is the resting membrane potential and EAP is
the threshold potential for initiating an action potential. In these animals,
the high efficiency of neuromuscular transmission is maintained during muscle
stretch. The safety factor for neuromuscular transmission does not change for
muscles at lengths between 80% and 120% of the resting length, because the
neuromuscular junction remains rigid so that the endplate membrane does not
deform when the muscle fiber changes length. The change in length can be
accommodated by folding and unfolding of the extrajunctional membrane, while
the endplate region remains rigid. Comparing the differences in the
transmission properties between the two kinds of animals, the physiological
importance of the force enhancement by stretch and its thermal acclimation in
poikilothermic animals are quite clear.
Effects of stretch on force in indirectly stimulated muscle
In the present study, enhancement of twitch force was not influenced by the
velocity of stretch but was critically dependent on the amplitude of stretch.
The enhancement appeared immediately after a stretch and was maintained. The
long-lasting enhancement is reminiscent of the well-known `residual force
enhancement after stretch' (Edman et al.,
1978,
1982
;
Edman and Tsuchiya, 1996
;
Herzog and Leonard, 2002
;
Herzog et al., 2003
) but the
effects observed in the present study are different from this, as explained
below.
Force enhancement effects that have not hitherto been reported are that a
muscle stretch applied during the silent period between twitches results in a
big force enhancement following the stretch, and this enhancement persists as
long as the muscle is elongated (Figs
4 and
7), whereas so-called `force
enhancement following a stretch' disappears when a muscle is deactivated
(Edman et al., 1978). These
differences suggest that the activation of muscle cells themselves is not
directly involved in the present mechanism.
We measured the electrical signals, because the conduction of electrical
signals between nerve and muscle seemed to be involved in the phenomenon. We
used suction electrodes (0.35 mm and 0.8 mm i.d. tip) for nerve and muscle,
respectively, to record action potential
(Fig. 10). The number of nerve
and muscle fibres thus measured by the electrode was approximately 17 and 8,
respectively, assuming that the average diameters of a nerve and a muscle
fibre are 20 µm and 100 µm, respectively. Therefore, the amplitude of
the action potential may reflect the total signals from fibres measured. Yepy
et al. (1974) mentioned that
the increased amplitude of the action potential in a compound muscle might be
due to the change in impedance of the muscle tissue upon stretch. But it may
be natural to interpret the result (Fig.
10) as an increase in the number of the activated muscle fibres
after stretch.
Effects of temperature on twitch force at various muscle lengths
One of the interesting results we observed is that twitch force in a
nerve-muscle preparation increased by stretch at short lengths and approached
the regression line of the length-force relationship measured on directly
stimulated muscle as length increased, before declining along the descending
limb of the relationship (Fig.
6). This effect was influenced remarkably by temperature. At high
temperature (22°C), 70-80% of muscle fibres were activated at resting
length (l0) without stretch and 20-30% could be readily
activated by a stretch (Fig.
6). Chen and Grinnell
(1997) showed that a change in
temperature had no measurable effects on the change in EPP amplitude and
frequency evoked by stretch between 13°C and 22°C. Their result seems
to be different from ours and the difference may arise from thermal
acclimation, discussed below.
Thermal acclimation in force development
It is known that events evoked at the neuromuscular junction by an action
potential of the nerve terminal, i.e. the release of neurotransmitter
substance, diffusion across the synaptic gap, and the response of the
end-plate on muscle, are all sensitive to temperature. Katz and Miledi
(1965) showed that the decay
phase of an action potential in the nerve terminal was lengthened, and
synaptic delay prolonged, at low temperatures. The amplitude of end-plate
current (EPC) was reduced at cooler temperatures
(Adams, 1989
;
Molgo and Van der Kloot,
1991
). These effects can be explained by a decrease in
highthreshold calcium channel activity of the nerve terminal, the reduced
affinity of the receptor on the end-plate, and the reduction in the number of
receptor channels available at low temperatures
(Janssen, 1992
). Thermal
acclimation at the nerve-muscle junction is possible in any part of the
pathways above-mentioned, but only a few effects have been reported so far.
The resistance of neuromuscular transmission to cold in the sartorius muscle
was increased when frogs were kept at low temperatures for prolonged periods
(Jensen, 1972
) and the latency
of the leg withdrawal reflex was shortened by cold acclimation
(Tiiska and Lagerspetz,
1999
).
In the present study, we measured the twitch force and its enhancement by
stretch at 4°C after the frogs had been kept at 4°C for more than 2
months and found that high twitch force was induced at the resting length
(l0) (Figs
8 and
9). Thus, the present
experiment clearly adds another example of thermal acclimation, based on our
experience in the present study. On transfer from room temperature to a low
temperature in the laboratory, immediately after the transfer frogs crawled
very slowly, but their behavior gradually livened up and after a few months at
the low temperature they could jump. Previous studies, by contrast, reported
that the mechanical performance of frog muscle was not different after
long-term exposure to different temperatures
(Renaud and Stevens, 1984;
Rome, 1983
). Thermal
acclimation of locomotory performance in frog is still a matter of controversy
(Brattstrom and Lawrence, 1962
;
Renaud and Stevens, 1983
;
Wilson and Franklin, 2000
;
Wilson et al., 2000
). The
frogs Rana brevipoda in the present study in Japan are active in a
warm environment, i.e. 14-27°C from April to October, and in hibernation
for the remaining months, which is different from the behaviour of the frogs
Rana temporaria and Rana pipiens used commonly in Europe and
America. Large variations in the sensitivity of thermal acclimation may exist
in animals in different geographic habitats
(Miller and Dehlinger, 1969
;
Wilson, 2001
).
Effects of temperature on twitch force
Our result showed that twitch force following indirect stimulation was
lower at lower temperatures (Fig.
3). The magnitude of twitch force in a whole muscle is the total
amount of twitch force produced by all fibres, and the number of recruited
muscle fibres for activation is smaller at lower temperature. There are
several possible reasons for this. (1) The decrease in twitch force at low
temperature is caused by the reduction in excitability of nerve that
innervates a muscle. This is not provable, however, because full tetanic force
was induced by indirect stimulation at low temperatures (Figs
2 and
3). (2) The reduction in the
efficiency of conduction at the neuromuscular junction at low temperature.
Previous investigators (Foldes et al.,
1978; Harris and Leach,
1968
) reported that the decrease in both release of acetylcholine
(ACh) and activity of acetyl cholinesterase (AChE) occurred together at low
temperatures, but the extent of the decrease in AChE activity was higher than
that of ACh release, resulting in a net decrease in the available ACh. Adams
(1989
) reported that the
proportion of fibres producing subthreshold EPC in response to a single nerve
shock was 42% at 5°C and 59% at 2.5°C. The proportion that we found
was approximately 88% at 4°C (Fig.
3). Thus our results, together with those of the previous report,
suggest that temperature strongly affects transmission at the neuromuscular
junction.
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Acknowledgments |
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References |
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