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Proc. R. Soc. B
doi:10.1098/rspb.2012.0467
Published online
Review
The mechanisms of the residual force
enhancement after stretch of skeletal
muscle: non-uniformity in half-sarcomeres
and stiffness of titin
Dilson E. Rassier*
Departments of Kinesiology and Physical Education, Physics and Physiology, McGill University,
475 Pine Avenue West, Montreal (PQ), Canada H4W 1S4
When activated skeletal muscles are stretched, the force increases significantly. After the stretch, the force
decreases and reaches a steady-state level that is higher than the force produced at the corresponding
length during purely isometric contractions. This phenomenon, referred to as residual force enhancement,
has been observed for more than 50 years, but the mechanism remains elusive, generating considerable
debate in the literature. This paper reviews studies performed with single muscle fibres, myofibrils and sarcomeres to investigate the mechanisms of the stretch-induced force enhancement. First, the paper summarizes
the characteristics of force enhancement and early hypotheses associated with non-uniformity of sarcomere
length. Then, it reviews new evidence suggesting that force enhancement can also be associated with sarcomeric structures. Finally, this paper proposes that force enhancement is caused by: (i) half-sarcomere
non-uniformities that will affect the levels of passive forces and overlap between myosin and actin filaments,
and (ii) a Ca2þ-induced stiffness of titin molecules. These mechanisms are compatible with most observations
in the literature, and can be tested directly with emerging technologies in the near future.
Keywords: force enhancement; cross-bridge kinetics; titin; sarcomere
1. INTRODUCTION
For many years, it has been known that skeletal muscles
which are stretched during activation present a significant
increase in force [1–4], while the energy consumption
decreases [5,6]. After the stretch, the force decays and
reaches a new steady state, which is higher than the force
produced at the corresponding length during purely
isometric contractions [1,2]. This increase in force has
been referred to as ‘residual force enhancement’, and
has been the focus of much research after the original
studies were published [7–12]. Incredibly enough, the cellular mechanism behind the residual force enhancement is
still elusive, and has been the topic of intense debate. This
form of force regulation is particularly challenging as it
cannot be readily explained by changes in overlap between
myosin and actin filaments and the classical force–length
relationship [13], and thus must be associated with
fundamental properties of muscle contraction.
In recent years, the emergence of new techniques has
allowed investigators to evaluate the force enhancement
with a high time and spatial resolution in isolated fibres,
myofibrils and single sarcomeres, which has brought new
insights into the phenomenon, ranging from the basic
characteristics to the proposed mechanisms. While some
of these studies focused on the residual force enhancement
[10,11,14], others aimed to evaluate the molecular aspects
of muscle contraction [15–18]. The latter studies still
added important data for a better understanding of the
mechanism of the force enhancement. This paper reviews
these new results in light of earlier studies and proposed
hypotheses, and suggests a testable mechanism for the
residual force enhancement, which is consistent with
most evidence from the literature.
2. CHARACTERISTICS AND NEW STUDIES OF THE
STRETCH-INDUCED FORCE ENHANCEMENT
There are many studies on the residual force enhancement,
with varying results and interpretations, at times contradictory. Since this review focuses on the mechanisms of force
enhancement, it will concentrate only on studies developed
with isolated muscle fibres, myofibrils or sarcomeres, in
which the average or individual sarcomere length was
measured or controlled during the experiments. Studies
with whole muscles and human subjects may offer insights
into potential physiological roles, but add little information
on the mechanisms of force enhancement.
A typical experiment showing residual force enhancement is shown in figure 1a. This experiment was
performed with an intact single fibre from the frog in the
laboratory of Dr Paul Edman—who has extensively investigated this phenomenon—and summarizes most of the
characteristics of the force enhancement. During the stretch
of an activated fibre, the force increases significantly. When
the stretch is performed at slow velocities (,2Lo s21), the
increase in force has two components. There is a sharp
increase in force that happens over approximately 9 nm
*[email protected]
Received 28 February 2012
Accepted 4 April 2012
1
This journal is q 2012 The Royal Society
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2 D. E. Rassier
Review. Mechanisms of muscle contraction
(a) 2.65
2.50
0·2 N mm–2
1s
stress (nN mm–2)
(b) 200
150
100
50
SL (mm)
0
3.0
2.8
2.6
0
2
4
6
8
time (s)
10
12
14
Figure 1. (a) Force enhancement after stretch of a single fibre
isolated from the frog anterior tibialis muscle. The figure
shows a stretch performed from 2.50 to 2.65 mm, and an isometric contraction developed at 2.65 mm. The force after
stretch remains higher than the force produced during the
isometric contraction. Adapted from Edman & Tsuchiya
[8] with permission. (b) Contractions produced by a single
sarcomere isolated from the rabbit psoas muscle, during an
experiment performed to evaluate force enhancement.
Initially, the sarcomere was activated isometrically in different lengths—in this case, after initial shortening the lengths
were approximately 2.7 mm (black line) and approximately
2.82 mm (green line). Then, the sarcomere was activated to
reach a sarcomere length of 2.7 mm and then it was stretched
to approximately 2.82 mm (red line). Similarly to the experiment with a single fibre shown in panel (a), the force
remains elevated after stretch. The figure also shows an isometric contraction produced after the stretch, performed at
the same length as in the beginning of the experiment, in
which the isometric force is similar (blue line), suggesting
the sarcomere was not damaged during the experiments.
per half-sarcomere [19,20]. Then, there is a slow phase in
which the force increases less steeply or remains unchanged
[7,8,19,21–23]. The transition between these phases is
associated with the mechanical detachment of cross-bridges
after they reach a critical extension [19,21,22,24]. The force
obtained at the transition point increases as a function of
stretch velocity to reach a maximum of approximately 2.0
Po at 1.0 mm s21 per half-sarcomere [20,21,23,25,26].
After the stretch, force decays in a bi-exponential fashion
(initially at 50 –250 s21 and then at 2–10 s21) [21,23,27]
before reaching a new steady state that is higher than the
force produced at the same length during purely isometric
contractions. The steady-state force enhancement is longlasting (.6 s), is enhanced with increasing amplitudes of
stretch [1,7] and initial sarcomere length [7], but is
independent of the speed of stretch [7,23].
Proc. R. Soc. B
In past years, myofibrils, which are preparations that
allow measurements of individual sarcomeres [28], have
been used to investigate the effects of stretch on force production [10,11,14,18]. These studies brought much
information to the field of muscle mechanics, but are
inconclusive and their interpretations vary largely. The
reason for such variability may be the inconsistency of the
experimental approaches used in different laboratories,
including a lack of repeatable control and isometric
contractions for comparisons with stretch contractions
(e.g. [14,29]), probably owing to the inherent difficulty of
performing mechanical measurements in myofibrils.
There is only one study, as far as this author knows, that
has investigated the residual force enhancement in myofibrils in a situation similar to what has been done in single
fibres [11]. The authors investigated small segments of
myofibrils, and observed force enhancement levels between
approximately 10 and 50 per cent, consistent with most
studies in the field (force enhancement ¼ steady-state
force after stretch/steady-state force during an isometric
contraction at the corresponding length). The same study
showed that force enhancement is also present in mechanically isolated sarcomeres at levels of approximately 10 per
cent [11]—a particularly important finding as it shows
that force enhancement cannot be entirely associated
with structural arrangements between sarcomeres in
series. The traces recorded during the experiments with
single sarcomeres are similar to those obtained with intact
fibre preparations (figures 1b and 2a). Interestingly and
new to the literature, the study also showed a population
of sarcomeres in which force enhancement was not present
after stretch (figure 2b), an observation with implications
for the mechanism of force enhancement, as will be discussed later in this review.
3. MECHANISMS OF FORCE ENHANCEMENT
The proposed mechanisms of force enhancement can
be classified into those involving: (i) sarcomere length
non-uniformities, or (ii) sarcomeric structures, including
(a) myosin cross-bridges and (b) passive elements, most
recently associated with titin molecules. These mechanisms are not necessarily mutually exclusive, as length
non-uniformities would affect contractile and passive properties of sarcomeres. It will be suggested later in this review
that force enhancement results from these mechanisms
working in parallel—the reason why several attempts to
isolate one mechanism have not been successful.
(a) Sarcomere length non-uniformity
It is well accepted that muscle fibres develop significant
sarcomere length non-uniformities upon activation
[9–11,28,30–34]. Non-uniformities are associated with
several experimental observations: the force creep in contractions produced at long sarcomere lengths [31,35],
differences in force obtained when fibres are activated
with sarcomere length control or fibre length control
[13,31,36], and the kinetics of force development and
relaxation [34,37,38]. Historically, sarcomere length nonuniformity has been incorporated into a hypothesis to
explain the residual force enhancement [39,40]. This
hypothesis has been a topic of much debate in the muscle
field, with heated discussions that extended from scholarly
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Review. Mechanisms of muscle contraction D. E. Rassier
50 nN mm–2
(b)
50 nN mm–2
(a)
2s
2s
(c)
sarcomere length (mm)
3
(d)
2.9
2.8
2.7
2.6
2s
(e)
2s
(f)
HS length (mm)
1.45
1.40
1.35
1.30
1s
1s
Figure 2. Experiments performed with one sarcomere isolated from the rabbit psoas muscle. (a,b) Force produced during an
isometric contraction (red) and a contraction in which the sarcomere was stretched (black). In the experiment shown in (a)
force enhancement was observed and in (b) force enhancement was not observed. (c,d) Sarcomere length changes during
the stretches (black) and the isometric (red) phases of the contractions. Note that upon relaxation the sarcomere stretches
slightly, returning to the pre-activation length. (e, f ) Half-sarcomere length changes during the activation and stretch (black)
or during isometric contraction (red). In both cases, the half-sarcomeres change length during the isometric period of
contraction.
publications to letters/responses to editors of scientific
journals [41–43].
According to this hypothesis, when muscle fibres are
stretched along the descending limb of the force– length
relationship, mechanical instability will lead to differences
in the yield tensions of sarcomeres, initiating a process
of length non-uniformities. As a result, sarcomeres will
stretch with varying lengthening velocities—the weakest
sarcomeres will lengthen very rapidly at the expense of
sarcomeres that will lengthen slowly. At one point, the
weak sarcomeres that are elongating rapidly will become
unable to hold the tension; they will lose all filament overlap and will ‘pop’, to be supported by passive forces. The
process will be repeated with all weakest sarcomeres,
which would also stretch beyond filament overlap to
stabilize higher force by means of passive forces.
Although first attempts to characterize the relation
between force enhancement and large non-uniformities
were not successful [7,12], the most important evidence
supporting this hypothesis was obtained in studies in
which muscles were frozen after stretch and analysed with
electron microscopy [44,45]. These studies showed sarcomere length non-uniformities in several sections of the
fibres, and striation patterns that suggest displacements of
Proc. R. Soc. B
the thick filaments relative to the centre of the sarcomeres,
between successive Z-lines [44,45]. Some sarcomeres
and half-sarcomeres were extended to long lengths, which
would support the notion of popping sarcomeres.
However, close analysis of the pictures in these papers
demonstrate that non-uniformities are scattered along
small sections of the frozen samples, and most importantly
sarcomeres elongated to a point in which they would lose all
filament overlap and pop are not evident—the reader is
encouraged to look at the pictures and judge.
In recent years, studies with isolated myofibrils and
single sarcomeres have cast more doubt into the popping
sarcomere hypothesis [10,11,28,34]. These experiments
confirmed that stretch in fact leads to the development
of sarcomere length non-uniformity, but without sarcomere popping—sarcomeres never stretched to the point
where they would lose all filament overlap [18,28].
Furthermore, force enhancement was observed on the
ascending limb and above the plateau of the force–
length relationship [10], which is inconsistent with the
idea of instabilities leading to popping sarcomeres.
In summary, activated muscle fibres and myofibrils
develop significant sarcomere-length non-uniformity upon
activation, which may increase significantly with an imposed
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4 D. E. Rassier
Review. Mechanisms of muscle contraction
stretch, but popping sarcomeres have never been observed.
Recently, such features and findings were extended to
suggest that force enhancement might be explained by
half-sarcomere length non-uniformities [11,18].
(i) Half-sarcomere length non-uniformities
Conceptually, non-uniformity in half-sarcomere lengths
induced at the beginning of activation would increase
throughout contractions, as the thick filaments would be
pulled towards the ends of the sarcomeres. Half-sarcomere
non-uniformities during isometric contractions have been
observed in myofibrils [18,34] and isolated sarcomeres
[46] (figure 2). In the case of single sarcomeres, A-band
displacements follow a characteristic pattern that resembles
the force–length relationship [46].
Can half-sarcomere length non-uniformities lead to a
residual force enhancement after stretch? Recently, a study
showed little correlation between force enhancement and
A-band displacements in single sarcomeres, but a very
strong relationship when experiments were conducted with
at least three sarcomeres in series [11] (figure 2). Again,
‘popping’ was not observed in half-sarcomeres, as they
never moved all the way towards the end of the sarcomeres,
staggering adjacent to the Z-lines, a finding in line with previous myofibrils studies that investigated this phenomenon in
a shorter time scale [18]. The maximal movement of A-band
displacements observed was approximately 50 nm per halfsarcomere, suggesting that some structure is preventing the
A-bands from moving towards the ends of sarcomeres.
Based on these findings, it seems likely that force enhancement has two components: (i) non-uniformity among
half-sarcomeres, and (ii) a sarcomeric component.
Half-sarcomere non-uniformity may lead to a complex
behaviour, as displacements of A-bands would result in variable amounts of filament overlap. There would be more
cross-bridges interacting with actin and thus more active
force production in strong half-sarcomeres. Titin filaments
would be overstretched and become stiffer in weaker halfsarcomeres, increasing the sarcomere strain and balancing
opposing forces in the strong halves. Such a mechanism
has been suggested in a different context by Edman and
Tsuchiya [8], but it could explain the length dependence
of the force enhancement. Force enhancement increases
when measurements are carried out at sarcomere lengths
up to approximately 20–30% longer than the plateau of
the force–length relation [7,8], a region where A-band displacement is significant [11] and passive forces start playing
a role in most skeletal muscles. Simultaneous with the
increase in passive strain and increase in filament overlap,
A-band displacements would cause cross-bridges to constantly stretch while the half-sarcomeres are not stabilized
[11,34,46], which could add to the force enhancement by
imposing resistance to the stretch—a phenomenon similar
to what happens during muscle fibre stretch.
A recently published computation model strengthens
the relation between force enhancement and halfsarcomere length non-uniformity [47]. In the model,
the authors show that the development of a small
degree of half-sarcomere heterogeneity causes force
enhancement at levels close to those observed in most
published experiments (approx. 5– 13%). The model
also predicts that force enhancement is dependent on
the stretch magnitude but independent of the stretch
velocity, consistent with findings in the literature.
Proc. R. Soc. B
(b) Sarcomeric structures
Since: (a) force enhancement is observed in sarcomeres
that do not show A-band displacements after stretch
and in situations where variability of sarcomere lengths
is small [10,11], and (b) the development of halfsarcomere non-uniformities (when present) induces
changes in the properties of sarcomeric structures, there
must be a mechanism of force enhancement that is contained within the half-sarcomere. The mechanism must
be associated with: (a) cross-bridge kinetics, and/or
(b) passive elements, more specifically titin molecules.
(i) Myosin cross-bridges
During the stretch of muscle fibres, there is an increase in
fibre stiffness, a putative measurement of the number of
cross-bridges attached to actin. The levels of increase in
stiffness vary between 10 and 60 per cent [19,20, 48,49],
raising controversy over the actual involvement of crossbridges. Some authors suggest that the increase in force is
caused by an increased mean force produced by the
cross-bridges [50,51] and a redistribution of cross-bridges
between pre-powerstroke and post-powerstroke states
[19,22,23,26,52]. Others suggest the increase in force is
caused by an increase in the number of cross-bridges
attached to actin [49,53], which may include the involvement of a second, adjacent cross-bridge that shares the
same myosin neck fragment. Regardless of the actual mechanism, the increase in stiffness vanishes quickly after
stretch, to levels between 0 and 7 per cent (between 10
and 300 ms after the stretch) [19,49], which weakens the
possibility that cross-bridges contribute to the residual
force enhancement.
Furthermore, studies that measured the stiffness during
the steady-state force enhancement period observed that it
decreased after stretch, reaching levels similar to those
observed during isometric contractions [9,12]. There is
one study that showed an increase in stiffness after stretch
when compared to isometric contractions produced at
corresponding lengths [16]. However, the increase in
stiffness was attributed to non-cross bridges structures
(see §3a(ii)).
Finally, a study showed that when myofibrils are activated with different levels of MgADP, which causes an
increase in the proportion of cross-bridges strongly
bound to actin, the levels of force enhancement were
comparable to myofibrils activated with Ca2þ [10],
strengthening the notion that force enhancement is not
associated with cross-bridge kinetics.
(ii) Passive elements and titin
In recent years, it has been suggested that force enhancement may be caused by non-cross-bridge, visco-elastic
structures and most specifically titin molecules [11,16,
23,26,54,55]. Titin spans the half-sarcomeres, attaching
to the Z-lines, thick and thin filaments, and acts as a
molecular spring providing most of the passive force in
muscle fibres in elongated sarcomeres. In skeletal
muscles, titin is present in one major N2A isoform,
which can however be expressed in different lengths; in
rabbit soleus the isoform is 3.7 MDa, and in rabbit
psoas it has 3.7 and 3.4 MDa forms [56]. According to
this hypothesis, when muscle fibres are activated, an
increase in the intracellular Ca2þ concentration (either
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Review. Mechanisms of muscle contraction D. E. Rassier
(iii) Mechanisms of titin regulation of force enhancement
The increase in passive forces with Ca2þ can hardly be
ignored when one evokes a mechanism of force enhancement after stretch. Obviously, it is important to understand
how titin regulates the increases in passive forces in the presence of Ca2þ after stretch. In this regard, there are two
proposed mechanisms: a direct effect of Ca2þ on titin
stiffness, or an effect of Ca2þ on the titin–actin interactions.
During activation, an elevation in intracellular Ca2þ
concentration can increase the stiffness of the Pro-GluVal-Lys (PEVK) element of titin—the region believed to
Proc. R. Soc. B
(a)
a
b
100 nm hs–1
29 kN m–2
c
d
100 ms
force (mN)
(b) 0.8
pCa2+ 4.5
0.6
0.4
pCa2+ 9.0
0.2
0
(c) 0.5
force (mN)
as a result of electrically induced action potentials in
intact fibres or changes in external Ca2þ concentrations
in permeabilized fibres) would trigger not only myosin –
actin interactions, but also a stiffening of titin molecules.
The increase in titin stiffness could increase force
considerably when the sarcomeres are stretched.
Although direct evidence that Ca2þ-induced ‘activation’ of titin is responsible for force enhancement
is still lacking, studies conducted independently in different laboratories provide data supporting this hypothesis.
The most significant results include the presence of stiffness in the sarcomere that is not associated with crossbridges—the static stiffness—and changes in the passive
force–sarcomere length relation with Ca2þ.
Studies have been reporting for a few years the presence of a ‘static’ stiffness and tension in skeletal muscle
fibres, which is activated by stretch [57 – 60] (figure 3a).
When intact muscle fibres are activated in the presence
of different myosin inhibitors and stretched, the stiffness
increases sharply, independently of cross-bridge attachment to actin. This static stiffness remains elevated for
as long as activation persists, i.e. a static tension is present
after stretch. The static tension has characteristics that are
similar to the residual force enhancement: it increases
with the amplitude of stretch and initial sarcomere
length, but is independent of the velocity of stretch
[57 – 59]. A recent study conducted with intact fibres isolated from the mouse showed that the static stiffness is
greater in digitorum longus (fast) muscle than in soleus
(slow) muscle [61]. The muscle type dependence
strengthens the possibility that static stiffness is caused
by titin, as the two muscles have different titin. Finally,
a study performed with permeabilized fibres from mammalian muscles, in which extracellular structures and
events associated with Ca2þ release are not involved in
stiffness or force measurements, confirmed the presence
of the static tension, which was directly associated with
the residual force enhancement [16]. In a few studies,
the static tension is observed even a few seconds after
activation stops [55,62,63], although the nature of this
persistent passive force enhancement, is unknown, and
it is not always observed.
Studies with single fibres in which myosin–actin interaction is inhibited with chemical interventions or with
depletion of troponin and thin filaments show that increasing Ca2þ concentrations causes an upward shift in the
sarcomere length–passive force relationship [16,17,63]
(figure 3b). The levels of increase in passive forces range
between 5 and 10 per cent, which is in line with the levels
of force enhancement observed in isolated sarcomeres
that do not show half-sarcomere length non-uniformities.
5
0.4
0.3
0.2
0.1
0
2.6
2.8
3.0
3.2
3.4
sarcomere length (mm)
3.6
Figure 3. (a) Force response to a stretch (amplitude 29.7 nm
per half-sarcomere, duration 1.2 ms) applied during the tetanus in a single fibre isolated from the frog anterior tibialis
muscle, treated with 2,3-butanedione monoxime solution
(6 mM). The picture shows: a, average sarcomere length;
b, tetanus with stretch; c, isometric tetanus; and d, the
force trace obtained by subtracting the passive force response
(not shown) and trace c from trace b. The ‘static tension’ was
measured after the end of the fast transient, as indicated by
the arrow. Adapted from Bagni et al. [57] with permission.
(b) Consecutive stretches performed with a permeabilized
fibre from the rabbit psoas muscle in pCa2þ 9.0 and 4.5.
An increase in Ca2þ concentration caused an increase in
forces produced by the fibre during and after stretch. The
passive force–sarcomere length relation in a series of experiments (n ¼ 7) performed before and after treatment of
fibres with rigor-EDTA, gelsolin and blebbistatin, in pCa2þ
9.0 (open symbols) and pCa2þ 4.5 (closed symbols). The
passive forces after the stretches increase at pCa2þ 4.5.
be Ca2þ sensitive. In a comprehensive study using different
fragments of titin, Labeit et al. [17] observed that Ca2þ
binding to the PEVK region of the molecules caused a
decrease in its persistence length. A decrease in the persistence length is associated with an increase in stiffness, and
consequently passive force production. The authors also
showed that the minimal titin fragment that responded to
Ca2þ contained a central E-rich domain with glutamates
flanked by PEVK repeats. Since skeletal muscle titin isoforms contain a variable number of PEVK repeats and Erich motifs [64], their result is consistent with the idea
that Ca2þ affects the conformation of the PEVK segment.
As a result, when muscles are activated with Ca2þ and
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6 D. E. Rassier
Review. Mechanisms of muscle contraction
actin
myosin
(a)
PEVK
PEVK + Ca2+
titin
(b)
M-line
(c)
Figure 4. (a) Diagram representing the major components of the sarcomere and the proposed mechanism of force enhancement. During activation of the sarcomere, some regions of the PEVK domain of titin are stiffened by Ca2þ. (b) When the
activated sarcomere is stretched, titin is stretched and the Ca2þ-bound region of the PEVK domain (purple), which is stiffened,
produces a small amount of force enhancement. In cases where titin is not responsive to Ca2þ (i.e. different lengths of titin
isoform), there is no force enhancement. (c) In some cases, especially when there are sarcomeres in series, stretch will lead
to half-sarcomere length non-uniformity. In this case, one half of the sarcomere will have an increased filament overlap, and
the other half will maintain the force by stretching of titin, which added to an increased stiffness caused by Ca2þ binding
will equilibrate the sarcomere I bands to produce the force enhancement.
then stretched, the response of titin would be enhanced
when compared to stretch without the presence of Ca2þ.
Another mechanism by which Ca2þ could regulate the
influence of titin is by increasing its binding to actin,
which would increase the overall sarcomere stiffness. This
hypothesis is tempting, given the proximity between titin
and actin filaments in the I-band of the sarcomeres
[65–68] and the malleability of the PEVK domain of
titin, which may transit among different conformational
states [69] and bind F-actin [70–73]. It has been shown
that the binding of the PEVK domain of titin to actin can
be modulated by S100A1, a member of the S100 family
of EF-hand Ca2þ-binding proteins [73] which is present
at high concentrations in striated muscles [74].
While one study showed that titin inhibited significantly the sliding of the actin filaments on in vitro
Proc. R. Soc. B
motility assays in the presence of Ca2þ [65], subsequent
studies using recombinant titin fragments failed to
detect binding between the tandem immunoglobulin segments of titin and actin [70,73]. In fact, in one of these
studies it was suggested that S100A1 – PEVK binding
alleviates the PEVK-based inhibition of F-actin motility,
inhibiting PEVK – actin interaction and providing the sarcomere with a mechanism to free the thin filament from
titin before contraction, thereby reducing the titin-based
force. This latest finding confirmed speculations of
Stuyvers et al. [75], who demonstrated that the titin –
actin interactions-based stiffness of rat cardiac trabeculae
increases when Ca2þ levels decay during relaxation, opposite to what would have been expected if Ca2þ were to
increase the titin –actin stiffness. At the cellular level, a
recent study has compared muscle fibres treated for
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Review. Mechanisms of muscle contraction D. E. Rassier
removal of troponin C with fibres treated for the removal
of actin [16]. The two treatments produced similar
results: an increase in static stiffness and tension that
was directly associated with low levels of force enhancement, suggesting that the increase in force is associated
with changes in titin, and not titin –actin interactions.
In conclusion, force enhancement is caused partially
by an increase in titin stiffness, a process that is regulated
by binding of Ca2þ to the PEVK domain of the molecule,
independently of titin –actin interactions.
(iv) Other mechanisms
Other structural proteins may influence the levels of
force enhancement after stretch. Nebulin (approx. 700–
800 kDa) is a protein that spans the entire thin filament
and attaches to the Z-line. Nebulin acts as a ruler to maintain the length of the thin filament, and new evidence shows
that it also regulates myosin –actin interactions during contractions [76]. If stretch induces conformational changes in
nebulin, it could lead to an increased number of myosin–
actin interactions. Since an increase in stiffness after stretch
is not commonly observed (as explained earlier in this
review), the role of nebulin in force enhancement is not
likely, although it deserves investigation. Desmin (approx.
52 kDa) is a protein responsible for keeping adjacent myofibrils in register across the muscle fibre, through lateral
connections at the Z-discs, linking the contractile apparatus to the sarcolemma. If desmin filaments were strained
during and after stretch, they could increase the overall
stiffness of the sarcomeres, contributing to force enhancement in fibre preparations. Such a possibility also
deserves investigation, but since force enhancement is present in isolated sarcomeres and myofibrils, it could not be
explained entirely by a desmin-related mechanism.
4. SUMMARY OF THE PROPOSED MECHANISM FOR
FORCE ENHANCEMENT
Based on the evidence presented in the literature, this
review proposes that the mechanisms for the residual
force enhancement are associated with: (i) non-uniformity
among half-sarcomeres, which will influence both the stiffness of titin and the overlap between myosin and actin
in half-sarcomeres, and (ii) a Ca2þ-regulated increase in
stiffness during activation and stretch (figure 4). These
mechanisms represent a variation of the original nonuniformity hypothesis, but without popping sarcomeres.
It also includes the role of Ca2þ activation of titin, a recently
exploited phenomenon that may explain force enhancement in conditions in which sarcomeres are maintained
mostly uniform through activation and stretch.
These mechanisms explain the following experimental features observed in studies using single fibres,
isolated myofibrils and sarcomeres that used force and
sarcomere length measurements simultaneously and that
controlled the experiments for potential artefacts and
intra-sample variability:
— force enhancement is present in single fibres, myofibrils and sarcomeres if titin isoforms are responsive
to Ca2þ. Since there are different lengths of the skeletal muscle titin isoforms, there may be cases where
force enhancement is absent in isolated sarcomeres;
Proc. R. Soc. B
7
— the level of force enhancement in single sarcomeres
that do not present half-sarcomere length nonuniformities and are dependent on a Ca2þ-induced
increase in titin stiffness is smaller than preparations
presenting large levels of half-sarcomere length
non-uniformities;
— the level of force enhancement is dependent on the
magnitude of stretch and initial sarcomere length,
but not on the velocity of stretch;
— force enhancement can be observed along the ascending limb and plateau of the force–length relationship if
some half-sarcomeres increase filament overlap after
stretch and a Ca2þ-induced stiffer titin supports the
passive forces at the other halves (which may be at
the descending limb of the force– length relation); and
— force enhancement is present and enhanced by sarcomere length non-uniformities without the occurrence
of popping sarcomeres. Sarcomeres may present
considerably different lengths after stretch without
showing large mechanical instabilities [28,33].
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