Force enhancement in single skeletal muscle fibres on the ascending limb of the forcelength relationship
University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4
* Author for correspondence (e-mail: walter{at}kin.ucalgary.ca)
Accepted 17 May 2004
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Summary |
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Key words: force enhancement, muscle mechanics, single fibre, striated muscle, cross-bridge theory, force production, frog, Rana pipiens
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Introduction |
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Force enhancement is known to increase with increasing magnitudes of
stretch (Abbott and Aubert,
1952; Edman et al.,
1978
,
1982
), but is independent, or
at least very insensitive, to the speed of stretch (Edman et al.,
1978
,
1982
;
Sugi and Tsuchiya, 1988
). One
of the crucial ideas regarding the mechanisms associated with residual force
enhancement following stretch has been the suggestion that force enhancement
does not occur in regions where the active isometric force increases with
increasing muscle/fibre length; i.e. the so-called ascending limb of the
forcelength relationship (Edman et al.,
1978
,
1982
;
Julian and Morgan, 1979b
).
Rather, force enhancement has been thought to occur only on the so-called
descending limb of the forcelength relationship, a region in which the
active isometric force decreases with increasing muscle/fibre length
(Abbott and Aubert, 1952
; Edman
et al., 1978
,
1982
;
Julian and Morgan, 1979b
;
Herzog and Leonard, 2000
), and
in which sarcomere lengths are assumed to be unstable
(Hill, 1953
) and non-uniform
in length (Julian and Morgan,
1979b
; Morgan,
1990
,
1994
;
Allinger et al., 1996
).
Therefore, force enhancement has been associated frequently with the
occurrence of instability and the associated non-uniformities in sarcomere
lengths (Julian and Morgan,
1979a
,b
;
Edman et al., 1982
; Morgan,
1990
,
1994
;
Edman and Tsuchiya, 1996
;
Morgan et al., 2000
).
According to the sarcomere length non-uniformity theory, force enhancement
can only occur on the descending, but not the ascending limb of the
forcelength relationship for two reasons: (i) the ascending limb has a
positive forcelength slope, therefore sarcomere lengths are stable and
non-uniformities cannot occur, and (ii) even if sarcomere length
non-uniformities occurred on the ascending limb (i.e. some sarcomeres would be
shorter and some longer than the average length), the sarcomeres that would be
shorter than average would have a decreased force potential compared to the
average sarcomere length (Gordon et al.,
1966). Therefore, only a decrease, but not an increase, in force
could result from sarcomere length non-uniformities on the ascending limb of
the forcelength relationship.
Recently, Herzog and Leonard
(2002) showed a small, but
consistent force enhancement on the ascending portion of the
forcelength relationship in the cat soleus. However, their result may
have been caused by a few fibres of the whole muscle that might have been on
the descending limb of the forcelength relationship, while the whole
muscle exhibited properties associated with the ascending limb of the
forcelength relationship. In order to answer the question of whether
force enhancement can occur on the ascending portion of the forcelength
relationship, experiments on the single fibre level were needed. Thus, the
purpose of this study was to investigate the residual, steady-state force
enhancement following active stretch in single fibres of frog on the ascending
limb of the forcelength relationship. Based on results obtained with
whole muscle, we hypothesized that there would be a small but consistent force
enhancement on the ascending, positive slope, of the forcelength
relationship, provided that stretch conditions were optimized. If so, the
steady-state forces following stretch should exceed the purely isometric
forces at optimal fibre length, if the fibre was stretched from some initial
length on the ascending limb of the forcelength relationship to the
optimal fibre length.
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Materials and methods |
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Experimental setup
The tendons of the dissected fibres were gripped with small T-shaped pieces
of aluminum foil as close to the fibre as possible. The fibres were mounted in
an experimental chamber between a force transducer (Sensonor AE801, SensorOne
Technologies Corp., Sausalito, CA, USA) and a servomotor length controller
(Aurora Scientific, Aurora, ON, Canada). The chamber was filled with Ringer's
solution (NaCl 115 mmol, KCl 3 mmol, CaCl2 3 mmol,
NaH2PO4 2 mmol, NaHCO3 20 mmol, pH 7.5). The
temperature was controlled at 8°C (range: 610°C) during
all experiments.
Stimulation (Grass S88, Grass Instruments, West Warwick, RI, USA) was given through two parallel platinum wire electrodes on either side of the fibre mounted in the experimental chamber. Square pulses (0.4 ms duration) were delivered with an amplitude 25% above the voltage that produced maximal force (range: 5090 V). Each fibre was tested individually to induce a completely fused tetanic contraction with the lowest stimulation frequency possible (range: 2326 Hz).
Initial procedures
After determining the optimal voltage for stimulation, the fibre was left
to twitch every 90 s for at least 30 min. The fibre was then visually
inspected for signs of damage. The magnitudes of the twitches after the
conditioning period were compared to the initial twitch values to examine the
quality and viability of the fibre. If any decrease in force was observed, the
fibre was discarded at this point.
Experimental procedures
An experimental forcelength curve was determined for each fibre
using 2 s contractions to define the ascending limb, the optimal length, and
the descending limb of the forcelength relationship
(Gordon et al., 1966;
Morgan et al., 2000
). The
length at which the greatest active force was obtained was defined as 0%
(optimal length), and was associated with an average sarcomere length of 2.1
µm in accordance with results of length-clamped
(Gordon et al., 1966
) and
fixed-end single fibre preparations (Lutz
and Rome, 1994
). Lengths below 0% were defined as the ascending
limb, and were designated with negative length values (i.e. a length of
10% refers to a fibre length that is 10% smaller than optimal length),
and the corresponding average sarcomere lengths were assumed to change
proportionally with fibre length. A rest period of 56 min was given
between contractions to avoid fatigue of the fibre. For the remainder of the
experiment, isometric reference contractions at the optimal length were
systematically used to check if force remained constant throughout testing. If
the force dropped more than 2% between adjacent reference contractions, or if
the reference force decreased by a total of 10% or more of the initial maximum
isometric force at any point during testing, the fibre was immediately
discarded, and the results were eliminated from analysis.
After determining the forcelength relationship and the optimal
length, isometric contractions (square pulses, 0.4 ms duration, 6 s
stimulation train), were performed at lengths of 0%, 5%, 10%,
15% and 20%; i.e. on the ascending limb of the
forcelength relationship (average sarcomere length of approximately
2.1, 2.0, 1.9, 1.8 and 1.7 µm, respectively). Then, three active stretches
were performed along the ascending limb of the forcelength relationship
from lengths of 10% to 0% (optimal length), 15% to 5%,
and 20% to 10% (Fig.
1). For the stretch tests, fibres were shortened passively to the
initial length (Li), where they were stimulated for 1 s,
which was sufficient time for the force to reach maximal isometric force (e.g.
Edman et al., 1978,
1982
;
Julian and Morgan, 1979b
;
Edman and Tsuchiya, 1996
).
Fibres were then stretched to the final length (Lf) at a
speed of 40% of fibre length s1 (i.e. in 0.25 s), where they
were held activated for another 4.75 s
(Fig. 1).
|
Data analysis and statistics
The active forces recorded at 3.75 s after the stretch were used for
statistical analysis, when force had reached a steady-state level (i.e. the
stretch test forcetime traces were parallel to the isometric reference
forcetime traces, e.g. Edman et al.,
1982). Parallelism was evaluated by fitting a linear regression
line through the data points for the 0.5 s period preceding the instant at
which force enhancement was evaluated. The slopes for both the isometric
reference and the stretch contractions were determined and evaluated for
differences using a repeated-measures analysis of variance (RM-ANOVA)
(P<0.05). Comparisons of forces at 3.75 s after stretch with the
isometric reference forces at the corresponding lengths were made by one-way
RM-ANOVA, followed by contrasts chosen a priori when indicated. A
level of significance of P<0.05 was used for all analyses. Results
are shown as means ± S.E.M.
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Results |
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In many cases, the steady-state forces following active stretch were greater than the isometric reference forces at optimal fibre length (Fig. 3). For the tests beginning at a length of 10% and finishing at 0% (optimal) length, all ten fibres showed force enhancement that exceeded the isometric force at optimal length by 4.9±0.9%, with a range of 0.810.9%. For the tests beginning at a length of 15%, eight out of the ten fibres showed force enhancement exceeding the force at optimal length. The mean force enhancement for these tests was 4.5±0.9%, and they exceeded the maximal isometric force by 2.4±1.0%. For the tests beginning at a length of 20%, the mean force enhancement was 2.0±0.6%, and the steady-state forces following active stretching never exceeded the purely isometric reference force at optimal length.
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Discussion |
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However, it has been shown that stretch-induced, steady-state force in
single skeletal muscle fibres can exceed the isometric force at optimal
length. Herzog and Leonard
(2002) and Rassier et al.
(2003
) found evidence for the
recruitment of a passive elastic element that contributed to the force
enhancement. They suggested that possibly this `passive force enhancement' may
account for the steady-state forces above the isometric reference force at
optimal fibre lengths. In the present study, no passive forces were observed
in any test, and no passive force enhancement either. However, this does not
preclude that passive force enhancement may cause the steady-state isometric
forces following stretch to exceed the purely isometric forces at optimal
length. In contrast to long muscle/fibre lengths, the passive force
enhancement at short lengths would have to disappear in this scenario, and
thus, would not be directly observable.
Edman et al. (1978) showed
force enhancement above the forces obtained at optimal length for stretch
magnitudes of 0.2 µm per sarcomere (about 10% of fibre length) in their
single fibres from frog (Rana temporaria) semitendinosus (their
figure 4A). However, they argued that this force enhancement was transient and
decayed rapidly, and was not visible if the fibre was held for 4.5 s following
the stretch (their figure 6A). However, the evidence for this latter statement
was based on a stretch of 0.08 µm per sarcomere, which corresponds to only
about 4% of fibre length. Therefore, it seems quite possible that Edman et al.
(1978
) might have found similar
results as shown here, had they performed systematic stretch experiments with
greater stretch amplitudes on the ascending limb of the forcelength
relationship.
There are a number of studies in which residual force enhancement has been
observed following stretch of whole muscles
(Abbott and Aubert, 1952;
Herzog and Leonard, 2000
,
2002
;
Herzog et al., 2003
). However,
the observed force enhancement may have been caused by some fibres that were
already on the descending portion of the forcelength relationship,
while the majority of fibres were still on the ascending limb. This scenario
would be associated with ascending limb behaviour of the whole muscle, and
simultaneously explain why force enhancement was relatively small in these
studies. Here, we observed small, but consistent, force enhancement on the
ascending limb of the forcelength relationship in single fibres. This
result implies that sarcomere length non-uniformities may develop on the
ascending portion of the forcelength relationship, in contrast to what
has been found experimentally (Julian and
Morgan, 1979b
; Morgan,
1990
,
1994
), and has been predicted
theoretically (Allinger et al.,
1996
).
The mean force enhancement (about 35%, with peak values reaching
about 10%) for the stretch protocols used here are much smaller than what has
typically been observed on the descending limb of the forcelength
relationship (Abbott and Aubert,
1952; Edman et al.,
1978
,
1982
;
Julian and Morgan, 1979b
;
Herzog and Leonard, 2000
).
Thus, it might be argued that force enhancement on the ascending limb is not
important for practical applications. However, from a mechanistic point of
view, the results of this study are significant, because they show that
systematic force enhancement is obtained at fibre lengths at which sarcomere
length non-uniformity cannot contribute to the total force enhancement.
Therefore, force enhancement may be associated with yet another mechanism that
has not been considered in the past, or might be caused by passive elements
whose effect disappears with deactivation in short, but not long, muscle
fibres.
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Conclusions |
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References |
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