| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SKELETAL MUSCLE AND EXERCISE |
1 Laboratory for Biomechanics, ETH Zürich, 8093 Zürich, Switzerland
2 Institute of Vegetative Physiology, University of Cologne, 50931 Cologne, Germany
3 Department of Physiology, University of Bristol, Bristol BS8 1TD, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
0.2 L0 s–1. The myofibril force response to a ramp stretch is similar to that reported from muscle fibres. Despite the 2.5-fold increase in force due to the stretch, the variability in half-sarcomere length remained almost constant during the stretch and A-band shifts did not progress further, independent of whether half-sarcomeres shortened or lengthened during the initial Ca2+ activation. Moreover, albeit half-sarcomeres lengthened to different extents during a stretch, rapid elongation of individual sarcomeres beyond filament overlap (‘popping’) was not observed. Thus, in contrast to predictions of the ‘popping sarcomere’ hypothesis, a stretch rather stabilizes the uniformity of half-sarcomere lengths and sarcomere symmetry. In general, the half-sarcomere length changes (dynamics) before and after stretch were slow and the dynamics after stretch were not readily predictable on the basis of the steady-state force–sarcomere length relation.
(Received 23 January 2006;
accepted after revision 7 March 2006;
first published online 9 March 2006)
Corresponding author J. Denoth: Laboratory for Biomechanics, ETH Zürich, ETH Hönggerberg, HCI E 357.1, CH-8093 Zürich, Switzerland. Email: jdenoth{at}ethz.ch
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
3.6 µm), so that the force produced by them would be transmitted entirely by a non-cross-bridge mechanism, the passive elasticity of the sarcomere. This rapid process of instability is often referred to as ‘sarcomere popping’. Meanwhile, the majority of the sarcomeres (‘strong’ sarcomeres) that lie in series would lengthen but less than expected if the stretch was uniformly distributed. After stretch the non-uniformity in sarcomere length yields a higher force compared with the end-held, isometric contraction at the corresponding longer mean sarcomere length (Julian & Morgan, 1979) since only few sarcomeres have effectively lengthened during stretch; the lengths of the majority of sarcomeres after stretch is overestimated, and final force is higher than expected from the mean sarcomere length (Morgan, 1994). Other researchers (Edman & Tsuchiya, 1996) argued that, due to redistribution of lengths after an imposed stretch, the ‘strong’ sarcomeres can operate at their full force-generating capacity by slightly shortening and acquiring a greater amount of filament overlap. In a later study Talbot & Morgan (1996) reported some electron micrographs of fixed fibres that indirectly supported their claim. However, shock-freezing or rapid fixation of the fibres may enhance disordering of sarcomeric structures. The validity of the sarcomere popping mechanism has been questioned by experiments that showed that all segments of a muscle fibre lengthen during stretch (Hill, 1977; Edman et al. 1982). In a recent study Rassier et al. (2003a) presented lengths of individual sarcomeres during ramp stretch of activated myofibrils and concluded that sarcomeres were non-uniform but ‘stable’, implying that ‘popping’ did not occur. However, they did not monitor sarcomere lengths during the initial relaxed phase and during activation prior to stretch, and did not record force; this would be important to fully understand the behaviour of sarcomeres in terms of active and passive mechanical components. Furthermore, as we showed previously (Telley et al. 2006), the two halves of a sarcomere operate differently during contraction. Therefore, accurate measures of the filament sliding in each half-sarcomere are necessary to validate whether popping occurs at the level of the functional unit of striated muscle, i.e. the individual half-sarcomere.
To date, time-resolved length measurement of individual active half-sarcomeres during stretch has not been pursued to reveal rapid elongation and loss of overlap. A single isolated myofibril would be an ideal preparation for such experiments since its sarcomeres can be readily and directly visualized. Thus, we have examined by direct and high-resolution measurement the lengths of individual half-sarcomeres (hSL) from Ca2+-activated psoas myofibrils that were stretched by 15–20%
L0 (resting length) at moderately fast velocities (range 0.15–0.20 L0 s–1).
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Rabbits were killed by cervical dislocation followed by exsanguination, and experiments were approved by the local animal care committee of the University of Cologne. Myofibrils were isolated from skinned strips of the psoas muscle, and immunostained at their Z-lines with an anti-
-actinin (clone EA-53, Sigma) and at their M-band with an anti-myomesin antibody (Grove et al. 1984) conjugated with a fluorescently labelled (Alexa 488) secondary antibody (ZENON., Molecular Probes), as described in detail in Telley et al. (2006). The relaxing and activating solutions consisted of 10 mM imidazole (pre-adjusted to pH 7 by 6 mM HCl), either 3 mM EGTA + 6 mM KCl pre-adjusted to pH 7 by 6 mM KOH (relaxing solution) or 3 mM EGTA + 3 mM CaCl2 pre-adjusted to pH 7 by 12 mM KOH (activating solution), 1 mM Na2MgATP, 3 mM MgCl2, 37.7 mM disodium creatine phosphate, 30 mM potassium glutathione and 30 mM DTT, finally adjusted to pH 7.0 at 10°C and consisting of a final ionic strength (µ) of 170 mM.
Apparatus and experimental protocol
Details of the set-up, mounting of the myofibrils, force measurements and half-sarcomere length measurement have been previously described (Stehle et al. 2002; Telley et al. 2006). All experiments were performed at 10°C. The myofibril was mounted in relaxing solution between the tip of a piezo-driven tungsten needle and the adhesive-coated tip of an atomic force cantilever (stiffness, 2.8 µN µm–1). The average slack sarcomere length and the diameter of the myofibril were determined under phase contrast and bright field microscopy using appropriate filters to prevent early photo bleaching. The myofibril was then Ca2+ activated by changing rapidly (within 
10 ms) from relaxing (pCa 7.5) to activating solution (pCa 4.5). After Ca2+-induced force development, a ramp stretch (15–20%
L0) was imposed on the myofibril for 1 s by the piezoactuator (P842.20 Physik Instrumente, Waldbronn, Germany), followed by a hold period of the same duration. The myofibril was then relaxed by changing back to relaxing solution. Simultaneously to force recording, epi-fluorescence patterns of Z-lines and M-bands were recorded by digital video microscopy (100 Hz frame rate) using a CCD camera (C8800-01C, Hamamatsu Photonics, Herrsching, Germany).
Data analysis and definitions
Video streams were visually inspected and post-processed to obtain individual half-sarcomere lengths and A-band shifts. Epi-fluorescence patterns were localized with a region-based tracking algorithm described in Telley et al. (2006). Half-sarcomere length (hSL) was defined as the distance between centres of the M-band and Z-line patterns (M–Z distance), and A-band displacement (
L) as the distance between the sarcomere centre (half of the Z–Z distance) and the M-band position. The accuracy of absolute initial half-sarcomere lengths measured using image segmentation was 35 nm. The accuracy of length changes during localization from one image to the next (‘tracking’) was 15 nm and dependent on image quality (signal-to-noise ratio).
Unless mentioned otherwise, a half-sarcomere (hS) is referred to as ‘weak’ or ‘strong’ when, upon Ca2+ activation prior to the stretch, it shortened less than or more than mean sarcomere length, respectively. This definition is meaningful and convenient for our data presentation but it is not exactly the same as that adopted on the basis of steady-state force versus sarcomere length relation (F–SL relation; see Discussion).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1.2 µm (sarcomere length, SL 2.4 µm), which is near the beginning of the descending limb of the force–sarcomere length relation for mammalian muscle (Sosa et al. 1994; Edman, 2005). A total of 204 half-sarcomeres were monitored during this study.
|
Figure 2 illustrates experimental recordings from the myofibril shown in Fig. 1A. The myofibril was Ca2+ activated while its total length was being held constant. Upon activation, the force (dotted trace in Fig. 2A) rises in a mono-exponential manner to the plateau isometric tension, P0. The lengths of 21 half-sarcomeres and fluorescent spots close to the two attachments of the myofibril could be monitored throughout the experiment. The total length change of the whole myofibril shown in Fig. 2B was derived from the change of distance between the two fluorescent spots at the attachments. During Ca2+-induced force development, total length shortened by 1% (310 nm), partly due to the distortion of the cantilever (90 nm) and partly due to the series compliances at the attachments (220 nm).
|
20% in 1 s, followed by isometric hold at the stretched length (Fig. 2B). The force response due to the stretch shows the basic features expected to occur upon ramp stretch-and-hold for stretches of these velocities: during the ramp, the force increased continuously with only moderate curvature and reached 2.5 P0 at the end of the ramp. When the myofibril was held isometric at the stretched length, force decreased in at least two exponential phases: initially by a fast decay (rate constant 10 s–1) followed by a slower decay. The slower decay occurred at force levels considerably higher (
1.5 fold) than P0. However, after a hold period of 1 s the force signal was still transient. Thus, the steady-state force approached at the stretched length, and hence the residual force enhancement cannot be accurately estimated from our records as the post-stretch duration was set short to minimize possible loss in myofibrillar function by photo-toxicity. After holding the myofibril for 1 s at the stretched length, relaxation was induced by reducing [Ca2+] whereupon the force decayed rapidly.
The continuous / dashed lines in Fig. 2A show exemplarily the length changes of four individual, neighbouring half-sarcomeres during the experiment. As reported in our previous study (Telley et al. 2006), shortening traces of half-sarcomeres upon Ca2+ activation are approximately bi-exponential and their variability in amplitudes indicate the non-uniform shortening behaviour of individual half-sarcomeres. During force development, length changes in the remaining 18 half-sarcomeres, either shortening or some lengthening, compensated the large shortening of the middle half-sarcomeres. Importantly, half-sarcomeres elongated during stretch at a time course similar to that of hS 15 shown in Fig. 2A, but none of the 22 half-sarcomeres visible during contraction and stretch lengthened beyond 1.45 µm. Interestingly, after the ramp stretch, several individual half-sarcomeres did not return to the same initial dynamic state (shortening, isometric or lengthening) they had before the stretch. At the stretched length, some half-sarcomeres lengthen while others shorten, but all at slow velocities (0.05–0.2 µm s–1). Moreover, the half-sarcomere (hS 14) that shortened considerably on activation (from 1.1 µm to 0.9 µm), and hence could be interpreted as being a ‘strong’ half-sarcomere, was elongated by the others after the stretch, despite operating on the plateau of the force–sarcomere length relation (hS 1.05 µm). Another half-sarcomere (hS 15) that shortened much less than the former on activation (from 1.2 µm to 1.1 µm), hence a ‘weak’ half-sarcomere, shows strikingly similar elongation during and after the ramp as the strong half-sarcomere. Although by definition a ‘weak’ half-sarcomere, it shortened shortly after the ramp. In general, these observations imply complex hS dynamics.
Half-sarcomere dynamics during and after stretch
The myofibril depicted in Fig. 1B (52 half-sarcomeres) exhibited considerable non-uniform behaviour in its left (hS 1–16) and right (hS 45–52) end-regions – whereas a more uniform, predominantly shortening behaviour occurred in the middle region (hS 17–42). Figure 3 shows an overview of its force response (A), traces of the segment lengths (B) and sample traces of individual half-sarcomere length (C and D). Mean hSL of the myofibril prior to activation was set to 1.2 µm, but the length traces during the first 80 ms after Ca2+ application could not be analysed because of out of focus images. In Fig. 3B (see figure legend for details) the thick curve is the length of the whole myofibril, the dotted curve represents the mean length of the 52 functional hSs, the dashed curve is the mean length of the hS 11–36 in the middle with the shaded area being their S.E.M.
– an index of hSL non-uniformity. It is seen that during Ca2+-induced force development the mean hSL decreased while the non-uniformity (i.e. S.E.M. or the spread of hSL) increased 1.5-fold. During the ramp stretch the mean hSL increased and remained almost constant after the stretch. More interestingly, the force increased by 200% while the non-uniformity increased by only 25% during the ramp stretch. This impressively illustrates that sarcomere inhomogeneity is not a simple function of force per se; the findings rather indicate that changing from slow shortening to lengthening (stretch) stabilizes hS homogeneity.
|
6 µm) by 27% during the Ca2+-induced force development and then by a further 33% during the ramp stretch (Fig. 3A, lower panel). The overall shape of the length transient of these ends is basically as expected from the force transient; the slight time course mismatch between the two (e.g. after the stretch) suggests that the ends are not purely linear elastic. On average, the mean length of the distal half-sarcomeres, hS 1–10 and 37–52 (dashed–dotted transient, in Fig. 3B) remained almost constant during the pre-stretch and post-stretch phase while during stretch these distal segments were elongated more than the middle 26 half-sarcomeres. However, considerable non-uniformity in hSL was present in these distal segments during the whole time course (data not shown). Upon Ca2+ removal at the stretched length, the mean hSL in all segments approached the value expected for the lengthened myofibril, accompanied by increasing uniformity indicated by the decreasing S.E.M. in hS lengths.
Figure 3C shows the dynamics of two half-sarcomeres (hS 17 and hS 18) that are somewhat similar to those shown in Fig. 2. On the other hand, a surprising observation, shown in Fig. 3D, is that a ‘strong’ half-sarcomere (hS 15, because it initially shortened on activation) elongated to 1.35 µm (i.e. beyond the plateau) during stretch, but shortened again at the stretched length (post-stretch phase). It lengthened slightly at a short hSL of 1.0 µm during late force rise on activation (initial pre-stretch phase), and was taking up approximately double (32%
L0) of the relative length change during stretch, whereas its neighbour (hS 16) lengthened only 1/6 of the applied stretch (2.5%
L0). It is important to note that after the large elongation during the ramp, the half-sarcomere hS 15 did not further extend towards non-overlap, resulting in ‘popping’, where force is borne entirely passively. After stretch these neighbouring half-sarcomeres had a concurrent dynamics, the longer one ‘creeping’ back to plateau length, while the shorter one slowly elongated. The complex intersarcomeric dynamics shown here can hardly be observed in the mean response of the myofibril (a population of sarcomeres) as shown in Fig. 3B.
To statistically analyse whether stretch-induced hS dynamics during and after stretch depend on their previous dynamics and/or on hSL, we fitted the three phases (pre-stretch, stretch and post-stretch) of the length traces with linear regressions to obtain the initial hSL and the sliding velocity for each phase. Figure 4 shows scatter plots of the velocities and hSL from 26 half-sarcomeres in the more uniform part of the myofibril (hS 11–36). The pre-stretch velocity has a significant correlation with the initial hSL (Pearson's product-moment, P < 0.05, Fig. 4A). However, we note that an initial part of the hSL transients i.e. the first 80 ms after Ca2+ application was missing and could not be included in this analysis. Thus, the initial hSL of the pre-stretch phase reflects the hSL approached 80 ms after onset of contraction. The correlation of velocity with initial hSL for the pre-stretch phase therefore probably indicates that the more a half-sarcomere shortened during this early time (80 ms) upon activation, the further it continues to shorten in the remaining pre-stretch period. Hence, prior to stretch, half-sarcomeres showed consistent dynamics according to our definition of ‘strong’ and ‘weak’ half-sarcomeres (see Methods).
|
A-band displacement
The general observation that neighbouring half-sarcomeres (sharing an M-band) show different dynamics was further analysed by measuring A-band displacements. Figure 5 shows data from a myofibril where a complete experiment as above was done. The myofibril consisted of 13 sarcomeres and we analysed 12 consecutive of the 26 half-sarcomeres, located at the right half of the myofibril. The left-hand column of Fig. 5 (A, C, E and G) show length traces from four selected neighbouring hS pairs, each pair sharing an M-line (continuous: right M–Z distance; dashed: left M–Z distance) and half of the length of the corresponding sarcomere (half of Z–Z distance, dotted line). In the frames on the right-hand side (B, D, F and H), the difference between the dotted and the dashed trace in each graph of Fig. 5A, C, E and G, respectively, is plotted, representing the displacement of the A-band relative to the sarcomere centre (sarcomere asymmetry) in the corresponding sarcomere. During Ca2+-induced force development (0.5–1.5 s), sarcomeres 2, 3 and 5 developed considerable asymmetry as indicated by separation of their half-sarcomere length traces in the left-hand frames and shift away from zero position in the right-hand frames. During ramp stretch (1.5–2.5 s), the asymmetry is greatly stalled. The observations after stretch are somewhat variable in detail for the different sarcomeres but, essentially, despite the large increase of force to 2.8 P0 due to the ramp stretch, the asymmetry did not progress much further in those sarcomeres where it developed during activation.
|
35 nm s–1 during the pre-stretch period but it was reduced to 20 nm s–1 during stretch.
The reduction is almost significant (P < 0.06, paired t test, Fig. 6) indicating that lengthening arrests/stabilizes A-band shift. The velocity was increased in the post-stretch period, 27 nm s–1, but the difference is not significant (P > 0.1).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
0.2 L0 s–1) is about 1/6 that of the maximum shortening velocity in rabbit psoas at 10°C (1.2 L0 s–1) (Sun et al. 2001), hence a moderate stretch velocity. When the resting half-sarcomere length prior to activation was set to 1.2 µm (SL 2.4 µm), corresponding to the beginning of the descending limb of the force–length relation in mammalian muscle (Sosa et al. 1994), activation by a step increase in [Ca2+] caused shortening of half-sarcomeres with bi-exponential dynamics shown previously (Telley et al. 2006), but also lengthening of some sarcomeres and extension of the series compliance at the attachment sites. Although the whole myofibril shortens 2–3% (20–30 nm per hS) selected half-sarcomeres shorten up to 300 nm. A ramp stretch induced a force rise to a peak with intermediate change in curvature similar to the results reported from muscle fibres (Edman et al. 1978; Lombardi & Piazzesi, 1990; Getz et al. 1998). The peak forces (2–3 P0) in our experiments suggest that the stretch velocity is at or beyond the ‘yield point’ of the force lengthening–velocity relation (Katz, 1939). During stretch all half-sarcomeres lengthened but by markedly different extents, in a range of 5 to 200% of the imposed length change per half-sarcomere. A well-known concept to characterize and predict muscle force relative to muscle length is the steady-state force versus mean sarcomere length (F–SL) relation. The F–SL relation was first shown by experiments of Gordon et al. (1966) and confirmed in mammalian fibres (e.g. Edman, 2005). The same concept has also been used to predict the dynamic behaviour of individual sarcomeres, particularly during muscle lengthening (Morgan, 1990; Allinger et al. 1996; Zahalak, 1997; Schachar et al. 2002). The general prediction was an isometric behaviour at optimal length range with maximal plateau force and occurrence of lengthening instability on the descending limb of the F–SL relation. We propose that this conceptual inversion is invalid because it cannot be extrapolated to the level of individual half-sarcomeres. Our study clearly demonstrates that the F–SL concept, per se, fails to foresee the behaviour of individual, active half-sarcomeres during and after stretch. Sarcomere lengthening and shortening are observed on the ascending limb, at the plateau, and on the descending limb. The failure has two reasons: firstly, the F–SL relation is a steady state and hence a static concept and, secondly, it does not take full account of serially linked functional components (half-sarcomeres) as found in myofibrils and fibres. Such an in-series system has a high degree of freedom and cannot be adequately described with just two parameters (force and end-to-end length). Instead, to fully account for the half-sarcomere behaviour in our experiments, dynamic concepts for each half-sarcomere, such as cross-bridge kinetics to model transient force response, and variability in force capacity among half-sarcomeres need to be considered.
No evidence of ‘popping sarcomeres’
On the basis of the methodology adopted in our experiments (as summarized above), the conditions were appropriate to expect rapid, uncontrolled elongation beyond filament overlap in some (‘weak’) sarcomeres (‘sarcomere popping’, see introduction) when a myofibril is stretched, as formulated by Talbot & Morgan (1996). Moreover, it may be argued that, with reduced transverse connections of sarcomeres, e.g. by desmin, an isolated myofibril would be more susceptible to stretch-induced changes than a whole muscle fibre. However, by visual observation and – more significantly – by accurate measurement of individual half-sarcomere length, we never observed such an event. This was also reported by Rassier et al. (2003b) but, unlike in the present study, they did not analyse the dynamic pre-history of sarcomeres during force development prior to stretch that provides some information on individual sarcomeric force capacity. We analysed in detail the video images and force recordings from six myofibrils for any evidence of rapid sarcomere elongation beyond filament overlap during ramp stretch. However, no ‘sarcomere popping’ was ever observed in any of a total of 204 half-sarcomeres. Instead, our data suggest that, although lengthening of half-sarcomeres is non-uniform, and lengthening and shortening half-sarcomeres co-exist, half-sarcomere dynamics are damped and slow, especially during the isometric hold phase after a stretch (post-stretch phase). Even those half-sarcomeres that elongated much more than the majority, which could be interpreted as being ‘weak half-sarcomeres’ according to Morgan's definition (Julian & Morgan, 1979; Morgan, 1990), did not necessarily further, and rapidly elongate after the ramp stretch to sarcomere lengths beyond 3.0 µm. Such subsequent lengthening was proposed some time ago by the same group to explain residual force after stretch, as caused by a decrease in force capacity with increase of length on the descending limb of the force–length relation, referred to in some papers (see, e.g. Zahalak, 1997) as ‘negative stiffness’ for instability. We cannot completely exclude the possibility that a decrease in filament overlap (‘sarcomere popping’) might occur at a sub-myofibrillar level, as the studies of Brown & Hill (1991) suggest, but our findings suggest no such sarcomeric or half-sarcomeric instability at the myofibrillar level and we were unable to observe any sarcomere ‘popping’ with the resolution of the methods we used.
Half-sarcomere dynamics and residual force enhancement after stretch
Our results show dynamic and complex behaviour of half-sarcomeres during and after stretch in which short (0.9–1.0 µm) half-sarcomeres can shorten or lengthen and long (> 1.3 µm) half-sarcomeres can shorten back towards the plateau of force–length relation. The length changes after stretch are markedly slow (< 0.05 L0 s–1). Recently, we showed by modelling that an in-series connected half-sarcomere system containing a 5–15% variability in isometric force capacity exhibits similar slow dynamics after stretch (Telley et al. 2003). In the model, a half-sarcomere is represented by an active force-generating component obeying the steady-state force–length and force–velocity relations (and Ca2+ sensitivity), and a passive tension component (representing titin) with non-linear viscoelastic properties. The novelty of the model was not in a new mechanism of active or passive force generation, but in a systematic treatment of the half-sarcomeres mechanically connected in series. The simulations presented there illustrated that half-sarcomere non-uniformity with slow dynamics is a natural response of a system having small gradients in active force capacity; this may arise from small differences in filament length, from disparities in filament spacing (Brown & Hill, 1991), etc. Such a possibility was indeed considered to overcome force oscillations in the modelled cross-bridge force response to stretch in muscle fibres (see discussion in Lombardi & Piazzesi, 1990).
Our multi-segmental modelling, shown in Fig. 7, illustrates a number of features of interest. Firstly, elongation during stretch is not necessarily restricted to a few half-sarcomeres and this does not end beyond filament overlap (SL > 3.5 µm). Secondly, a stretch on the plateau of the force–length relation can cause forces higher than P0 and force capacity of the strongest half-sarcomere. Thirdly, without occurrence of ‘sarcomere popping’, the internal slow dynamics after stretch can lead to residual force enhancement up to 10% P0. However, recruitment of some ‘sarcomeric structure(s) outside the cross-bridges’ on activation and during stretch, as indicated by the ‘static stiffness’ reported by Bagni et al. (2002), may need to be considered to account for the full extent of force enhancement.
|
10% P0 above the expected value at the final length. In contradiction to our modelling, the same group ruled out sarcomere inhomogeneity as a contributory mechanism for the residual force enhancement after stretch by arguing that (a) steady force after stretch should not exceed isometric force at optimum length (Rassier et al. 2003c), and (b) force enhancement should not occur on the plateau and ascending limb (Herzog & Leonard, 2002). Our experimental results show that during activation prior to stretch the sarcomere length distribution is already dispersed so that long (hSL 1.5 µm, mostly at the periphery) and short (hSL 0.9 µm) half-sarcomeres co-exist (present study and Telley et al. 2006) and predictions on the basis of the steady-state F–SL relation for the whole population are not meaningful. This is further supported by our modelling which demonstrates that ‘isometric’ force can well be exceeded during the hold period after stretch. Thus, contrary to what is suggested by Rassier & Herzog (2004), sarcomere non-uniformity, without sarcomere ‘popping’, does play a role in the residual force enhancement. Sarcomere asymmetry: coupling between active/passive forces in sarcomeres
Although they used long lasting contractions (duration of several minutes), Horowits & Podolsky (1987) were the first to provide evidence of A-band shift in contracting muscle fibres, resulting in asymmetry of filament overlap in the two halves of the sarcomere. Recently, we showed that such displacements indeed can occur during the first few seconds of contraction in end-held myofibrils (Telley et al. 2006), obviously caused by an imbalance in the force generation in the halves of a sarcomere. Other findings have indicated the importance and/or involvement of active and passive force transmission within sarcomeres in overall muscle function. For instance, Mutungi & Ranatunga (1996) observed that fast–slow muscle difference in the viscoelastic relaxation rate of resting tension (fast/slow ratio of 3) was similar to the difference in their active shortening velocity; they suggested that this is indicative of appropriate coupling/interaction between active and passive force-transmitting mechanisms within muscle.
The present study demonstrates that the A-band shift and sarcomeric asymmetry, that develop during the initial isometric contraction, remain during externally imposed force (stretches), albeit with small changes. During ramp stretch all half-sarcomeres lengthen; however, despite the development of high force due to the stretch, the increase in sarcomere asymmetry seen before stretch is clearly reduced during the stretch (see Fig. 6). The average dynamics of sarcomere asymmetry after the end of the stretch does not suggest a consistent behaviour, e.g. with simple predictions from the force–length relation. It may be argued that the A-band displacement is simply a phenomenon or outcome of sarcomeric stabilization, unbalanced cross-bridge force generation in the two halves of a sarcomere being compensated by stretching of passive components (e.g. titin) in the weaker half. An approximate estimation of force in a unit cell of the filament lattice in each side of the two halves in a sarcomere leads to the following: assuming three titin filaments and 150 potential cross-bridges on each half of the myosin filament, 40% occupation (Linari et al. 1998) and 8–10 pN isometric force per attached head (Piazzesi et al. 2002), a 10% difference in cross-bridge force between the two halves of a sarcomere would result in an imbalance of 50 pN in a unit cell. This would cause each titin filament in one half to transmit 9 pN more force than in the other. According to the measurements of Labeit et al. (2003) this would imply a length change of 50–100 nm in the compliant region of titin which is similar to the A-band shift observed in our experiments.
Taking the simplest case, two types of force can arise in an active half-sarcomere during lengthening. On the cross-bridge level, high force during stretch may arise from straining cross-bridges in a pre-power-stroke state which generates only low force under isometric conditions (Getz et al. 1998). Evidence implied by energy studies indicates that a stretch induces a truncation of the full cross-bridge cycle (Fenn, 1924; Abbott & Aubert, 1951; Curtin & Davis, 1973; Linari et al. 2003). On the level of ‘sarcomeric structure(s) outside the cross-bridges’, forces from stretching (and unfolding) the titin filament (Minajeva et al. 2001) may contribute considerably to force in lengthening muscle, even at shorter half-sarcomere lengths. Thus, the implication of our findings is that, under dynamic stretch conditions, titin may play an important role in force transmission across sarcomeres. However, purely elastic forces in titin at short half-sarcomere lengths are small and viscous forces could be a major contributor; indeed, moderately large velocity-sensitive, viscous-like force development occurs during stretch of relaxed muscle fibres, evidently from titin filaments (see Ranatunga, 2001). Additionally, titin stiffness may increase due to interaction with actin and due to Ca2+-induced stiffening on activation (Labeit et al. 2003). Significantly, our results show that the two force-developing components are less unbalanced between the halves of a sarcomere during lengthening than during active shortening (fully cycling cross-bridges). The finding that A-bands are stabilized during stretch is compatible with the fact that ‘popping’ sarcomeres were not observed. If selected half-sarcomeres were rapidly elongating, A-band displacements would then increase markedly, but such increases were never observed.
Conclusions
Our findings have raised a number of issues of interest. Firstly, the results provide no evidence in support of the ‘popping sarcomere’ hypothesis. Secondly, all half-sarcomeres lengthen during stretch and the steady-state force–sarcomere length relation, per se, does not predict the dynamic half-sarcomere behaviour, as used previously (e.g. Morgan, 1990). Thirdly, half-sarcomere dynamics after stretch are slow and not explainable without introducing variability in passive viscous components. Fourthly, development of sarcomeric asymmetry is generally reduced during stretch compared with that during initial isometric force development. Finally, it seems that simulation of the contractile behaviour of preparations consisting of a dozen or more functional elements (half-sarcomeres) in series requires the use of a multi-segmental model (instead of a simple lumped model) with assumptions of variability in their active and/or passive dynamic mechanical properties.
|
Footnotes |
|---|
Re-use of this article is permitted in accordance with the Creative Commons Deed, Atribution 2.5, which does not permit commercial exploitation.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Allinger TL, Epstein M & Herzog W (1996). Stability of muscle fibers on the descending limb of the force-length relation. A theoretical consideration. J Biomech 29, 627–633.[CrossRef][Medline]
Bagni
MA, Cecchi
G, Colombini
B
&
Colomo
F (2002). A non-cross-bridge stiffness in activated frog muscle fibers. Biophys J
82, 3118–3127.
Brown LM & Hill L (1991). Some observations on variations in filament overlap in tetanized muscle fibres and fibres stretched during a tetanus, detected in the electron microscope after rapid fixation. J Muscle Res Cell Motil 12, 171–182.[CrossRef][Medline]
Curtin NA & Davis RE (1973). Chemical and mechanical changes during stretching of activated frog skeletal muscle. Cold Spring Harb Symp Quant Biol 37, 619–626.
Edman
KA (2005). Contractile properties of mouse single muscle fibers, a comparison with amphibian muscle fibers. J Exp Biol
208, 1905–1913.
Edman
KA, Elzinga
G
&
Noble
MI (1978). Enhancement of mechanical performance by stretch during tetanic contractions of vertebrate skeletal muscle fibres. J Physiol
281, 139–155.
Edman
KA, Elzinga
G
&
Noble
MI (1982). Residual force enhancement after stretch of contracting frog single muscle fibers. J General Physiol
80, 769–784.
Edman KA & Tsuchiya T (1996). Strain of passive elements during force enhancement by stretch in frog muscle fibres. J Physiol 490, 191–205.[Medline]
Fenn
WO (1924). The relation between the work performed and the energy liberated in muscular contraction. J Physiol
58, 373–395.
Getz
EB, Cooke
R
&
Lehman
SL (1998). Phase transition in force during ramp stretches of skeletal muscle. Biophys J
75, 2971–2983.
Gordon
AM, Huxley
AF
&
Julian
FJ (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol
184, 170–192.
Grove
BK, Kurer
V, Lehner
C, Doetschman
TC, Perriard
JC
&
Eppenberger
HM (1984). A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies. J Cell Biol
98, 518–524.
Herzog
W
&
Leonard
TR (2002). Force enhancement following stretching of skeletal muscle: a new mechanism. J Exp Biol
205, 1275–1283.
Hill
L (1977). A-band length, striation spacing and tension change on stretch of active muscle. J Physiol
266, 677–685.
Horowits
R
&
Podolsky
RJ (1987). The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments. J Cell Biol
105, 2217–2223.
Julian
FJ
&
Morgan
DL (1979). The effect on tension of non-uniform distribution of length changes applied to frog muscle fibres. J Physiol
293, 379–392.
Katz
B (1939). The relationship between force and speed in muscular contraction. J Physiol
96, 45–64.
Labeit
D, Watanabe
K, Witt
C, Fujita
H, Wu
Y, Lahmers
S, Funck
T, Labeit
S
&
Granzier
H (2003). Calcium-dependent molecular spring elements in the giant protein titin. Proc Natl Acad Sci U S A
100, 13716–13721.
Linari
M, Dobbie
I, Reconditi
M, Koubassova
N, Irving
M, Piazzesi
G
&
Lombardi
V (1998). The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin. Biophys J
74, 2459–2473.
Linari
M, Woledge
RC
&
Curtin
NA (2003). Energy storage during stretch of active single fibres from frog skeletal muscle. J Physiol
548, 461–474.
Lombardi
V
&
Piazzesi
G (1990). The contractile response during steady lengthening of stimulated frog muscle fibres. J Physiol
431, 141–171.
Mansson A (1994). The tension response to stretch of intact skeletal muscle fibres of the frog at varied tonicity of the extracellular medium. J Muscle Res Cell Motil 15, 145–157.[CrossRef][Medline]
Minajeva
A, Kulke
M, Fernandez
JM
&
Linke
WA (2001). Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils. Biophys J
80, 1442–1451.
Morgan
DL (1990). New insights into the behavior of muscle during active lengthening. Biophys J
57, 209–221.
Morgan DL (1994). An explanation for residual increased tension in striated muscle after stretch during contraction. Exp Physiol 79, 831–838.[Medline]
Mutungi
G
&
Ranatunga
KW (1996). Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures. Biophys J
70, 1432–1438.
Noble MI (1992). Enhancement of mechanical performance of striated muscle by stretch during contraction. Exp Physiol 77, 539–552.[Medline]
Piazzesi
G
&
Lombardi
V (1995). A cross-bridge model that is able to explain mechanical and energetic properties of shortening muscle. Biophys J
68, 1966–1979.
Piazzesi
G, Lucii
L
&
Lombardi
V (2002). The size and the speed of the working stroke of muscle myosin and its dependence on the force. J Physiol
545, 145–151.
Ranatunga KW (2001). Sarcomeric visco-elasticity of chemically skinned skeletal muscle fibres of the rabbit at rest. J Muscle Res Cell Motil 22, 399–414.[CrossRef][Medline]
Rassier
DE
&
Herzog
W (2004). Active force inhibition and stretch-induced force enhancement in frog muscle treated with BDM. J Appl Physiol
97, 1395–1400.
Rassier DE, Herzog W & Pollack GH (2003a). Dynamics of individual sarcomeres during and after stretch in activated single myofibrils. Proc R Soc Lond B Biol Sci 270, 1735–1740.[Medline]
Rassier DE, Herzog W & Pollack GH (2003b). Stretch-induced force enhancement and stability of skeletal muscle myofibrils. Adv Exp Med Biol 538, 501–515.[Medline]
Rassier DE, Herzog W, Wakeling J & Syme DA (2003c). Stretch-induced, steady-state force enhancement in single skeletal muscle fibers exceeds the isometric force at optimum fiber length. J Biomech 36, 1309–1316.[CrossRef][Medline]
Schachar R, Herzog W & Leonard T (2002). Force enhancement above the initial isometric force on the descending limb of the force-length relationship. J Biomech 35, 1299–1306.[CrossRef][Medline]
Sosa
H, Popp
D, Ouyang
G
&
Huxley
HE (1994). Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths. Biophys J
67, 283–292.
Stehle R, Krüger M, Scherer P, Brixius K, Schwinger RH & Pfitzer G (2002). Isometric force kinetics upon rapid activation and relaxation of mouse, guinea pig and human heart muscle studied on the subcellular myofibrillar level. Basic Res Cardiol 97 (Suppl. 1), 127–135.
Sun
YB, Hilber
K
&
Irving
M (2001). Effect of active shortening on the rate of ATP utilisation by rabbit psoas muscle fibres. J Physiol
531, 781–791.
Talbot JA & Morgan DL (1996). Quantitative analysis of sarcomere non-uniformities in active muscle following a stretch. J Muscle Res Cell Motil 17, 261–268.[CrossRef][Medline]
Telley IA, Denoth J & Ranatunga KW (2003). Inter-sarcomere dynamics in muscle fibres. A neglected subject? Adv Exp Med Biol 538, 481–500.[Medline]
Telley
IA, Denoth
J, Stüssi
E, Pfitzer
G
&
Stehle
R (2006). Half-sarcomere dynamics in myofibrils during activation and relaxation studied by tracking fluorescent markers. Biophys J
90, 514–530.
Zahalak GI (1997). Can muscle fibers be stable on the descending limbs of their sarcomere length-tension relations? J Biomech 30, 1179–1182.[CrossRef][Medline]
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. Herzog and T. Leonard Reply from Walter Herzog (on behalf of the authors) and Tim Leonard J. Physiol., January 15, 2007; 578(2): 617 - 620. [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Rassier Stretching human muscles makes them stronger J Appl Physiol, January 1, 2007; 102(1): 5 - 6. [Full Text] [PDF]
|
|
|
|
|
|
D. L Morgan and U. Proske Sarcomere popping requires stretch over a range where total tension decreases with length J. Physiol., July 15, 2006; 574(2): 627 - 628. [Full Text] [PDF]
|
|
|
|
|
|
I. A. Telley, E. Stussi, J. Denoth, R. Stehle, G. Pfitzer, and K. W. Ranatunga Reply from I. A. Telley, R. Stehle, K. W. Ranatunga, G. Pfitzer, E. Stussi and J. Denoth J. Physiol., July 15, 2006; 574(2): 629 - 630. [Full Text] [PDF]
|
|
|
|
|
|
G. J. Pinniger, K. W. Ranatunga, and G. W. Offer Crossbridge and non-crossbridge contributions to tension in lengthening rat muscle: force-induced reversal of the power stroke J. Physiol., June 15, 2006; 573(3): 627 - 643. [Abstract] [Full Text] [PDF]
|
|
|
|
); a data pair is connected by a straight line to identify half-sarcomeres. The region between the vertical dotted lines (hSL = 1.0–1.2 µm) denotes the plateau region of the force versus half-sarcomere length relation. Note that all velocities are positive during stretch irrespective of the pre-stretch hSL (*): after the ramp stretch, the velocities are reduced to reach the longer hSL.
