Proc. R. Soc.

B (2012) 279, 981–990

doi:10.1098/rspb.2011.1304
Published online 7 September 2011

Is titin a ‘winding filament’? A new twist on

muscle contraction
Kiisa C. Nishikawa1,*, Jenna A. Monroy1, Theodore E. Uyeno2,
Sang Hoon Yeo3, Dinesh K. Pai3 and Stan L. Lindstedt1
1
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA
2
Department of Biology, Valdosta State University, Valdosta, GA 31698-0015, USA
3
Department of Computer Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Recent studies have demonstrated a role for the elastic protein titin in active muscle, but the mechanisms by
which titin plays this role remain to be elucidated. In active muscle, Ca2þ-binding has been shown to
increase titin stiffness, but the observed increase is too small to explain the increased stiffness of parallel
elastic elements upon muscle activation. We propose a ‘winding filament’ mechanism for titin’s role in
active muscle. First, we hypothesize that Ca2þ-dependent binding of titin’s N2A region to thin filaments
increases titin stiffness by preventing low-force straightening of proximal immunoglobulin domains that
occurs during passive stretch. This mechanism explains the difference in length dependence of force
between skeletal myofibrils and cardiac myocytes. Second, we hypothesize that cross-bridges serve not
only as motors that pull thin filaments towards the M-line, but also as rotors that wind titin on the thin
filaments, storing elastic potential energy in PEVK during force development and active stretch. Energy
stored during force development can be recovered during active shortening. The winding filament hypoth-
esis accounts for force enhancement during stretch and force depression during shortening, and provides
testable predictions that will encourage new directions for research on mechanisms of muscle contraction.
Keywords: connectin; force enhancement; force depression; history dependence of force production;
thin filament rotation; titin – actin interactions

1. INTRODUCTION [14]. Current work focuses on titin’s roles in regulating

Since publication of the sliding filament theory [1,2], myofibrillar assembly [15] and cell signalling [16]. It
remarkable progress has been made in elucidating molecu- has been suggested that titin could function as a spring
lar mechanisms of muscle contraction [3,4]. Despite this not only in resting muscles, but also in active muscles
progress, explanations for several features of muscle func- [17 – 20]. However, as yet, no compelling mechanism
tion remain elusive [5], including enhancement of force has been offered for how titin could play such a role.
with stretch [6]; depression of force with shortening [6]; We first review the layout of titin within muscle sarco-
the low cost of force production during active stretch [7]; meres, the contribution of titin to muscle passive tension,
and the high thermodynamic efficiency of actively shorten- and recent studies that demonstrate a contribution of titin
ing muscle [8]. Efforts to explain these and other to force production in active muscle. Next, we develop a
observations led to modifications of the original theory two-step winding filament hypothesis, in which Ca2þ
[9] as well as development of alternative hypotheses [10]. influx ‘activates’ titin via Ca2þ-dependent binding of
We propose a new addition to the sliding filament– N2A to the thin filaments, which shortens and stiffens
swinging cross-bridge theory: a two-step ‘winding filament’ the titin spring, after which the cross-bridges not only
model of muscle sarcomeres, in which titin is ‘activated’ translate but also rotate the thin filaments, winding titin
mechanically by Ca2þ influx, and then is wound upon the upon them and storing elastic potential energy in
thin filaments by the cross-bridges, which not only translate PEVK. Finally, we discuss the implications of the winding
but also rotate the thin filaments. We will show that this filament hypothesis for understanding muscle behaviour.
hypothesis provides a cogent framework for understand-
ing force enhancement and depression, as well as other
unexplained observations of muscle physiology. 2. LAYOUT OF TITIN WITHIN MUSCLE
Although the existence of titin-like fibres was inferred SARCOMERES
in early structural studies [2], titin (also known as connec- At up to 4.2 MDa, titin is the largest known protein [21]
tin) was discovered more than 20 years after development and the third most abundant protein in striated muscle
of the sliding filament theory [11,12]. Development of the [22]. Titin spans an entire half-sarcomere (approx.
sliding filament –swinging cross-bridge theory proceeded 1 mm) from Z-disc to M-line [15]. Each titin molecule
without consideration of titin. Early studies of titin estab- (figure 1a) is associated with one thick filament in the
lished its roles in maintaining sarcomere integrity [13] A-band and one thin filament in the I-band [23]. In
and contributing to passive tension of striated muscles each half-sarcomere, each thick filament surrounds a
core of six titin filaments, and each thin filament interacts
with three titin filaments that radiate from neighbour-
* Author for correspondence (kiisa.nishikawa@nau.edu). ing thick filaments [24]. An inextensible end filament,

Received 22 June 2011

Accepted 17 August 2011 981 This journal is q 2011 The Royal Society
982 K. C. Nishikawa et al. Is titin a winding filament?

(a) I-band A-band

proximal PEVK distal thick filament

Z-disc tandem Ig N2A tandem Ig thin filament M-line
segment segment

(b)

(c)

N2A

Figure 1. (a) Schematic of a skeletal muscle half-sarcomere illustrating the layout of titin (yellow with a red N2A segment).
Each titin molecule is bound to the thin filaments (blue) in the I-band, and to the thick filaments (green) in the A-band.
Note that, for simplicity, thick filaments are illustrated as double-stranded, whereas in vertebrate skeletal muscle, they are
actually triple-stranded. The N2A segment is located between the proximal tandem Ig segment and the PEVK segment.
(b,c) Ca2þ-dependent binding of N2A to thin filaments. (b) Resting sarcomere at slack length at low Ca2þ concentration
(pCa ¼ 9). (c) Upon Ca2þ influx (pCa ¼ 4.5), N2A titin binds to the thin filament (blue), which shortens and stiffens the
titin spring in active sarcomeres.

composed of the distal immunoglobulin (Ig) domains of spring at low force, but as an enthalpic, or Hookean,
six titin molecules, emerges from the tip of each thick fila- spring at higher forces [14,31]. As sarcomeres are stretched
ment at the A/I junction [24]. Interaction among the Ig from a resting length of 2.1–3.5 mm, the length of PEVK
domains maintains the axial position of the end filament increases more than 10-fold [14]. At physiological sarco-
at the centre of the hexagonal array of thin filaments mere lengths, elongation of PEVK largely determines the
that surround each thick filament [24]. passive elasticity of skeletal myofibrils and cardiac myocytes
The carboxy terminus of titin is located at the M-line, [28,32]. The sarcomere length at which passive tension
where titin filaments from adjacent half-sarcomeres with increases varies with the titin isoform expressed in different
opposite polarity overlap [15]. In the A-band, titin is muscles [28].
composed of repeating fibronectin (FNIII) and Ig
domains, rendered inextensible by their tight association
with the thick filament [25]. In the I-band, titin is com- 4. IS THERE A ROLE FOR TITIN IN ACTIVE MUSCLE?
posed of proximal and distal tandem Ig segments that In active muscle fibres, Ca2þ influx increases tension and
flank the PEVK region. The proximal tandem Ig and stiffness of a non-cross-bridge structure, possibly titin
PEVK segments function as springs in series [14,26]. In [19]. In both PEVK fragments and single muscle fibres,
the Z-disc, titin is anchored to thin filaments, a-actinin, Labeit et al. [20] demonstrated not only that Ca2þ influx
and anti-parallel titin molecules from the adjacent sarco- increases titin stiffness, but also that increasing Ca2þ in
mere [27]. Overlap of titin molecules in both Z-discs and myofibrils results in a leftward shift of the force–extension
M-lines produces a titin filament system that is continu- curve. However, the effects of Ca2þ on titin stiffness
ous along the entire length of every myofibril [15]. observed in these studies are approximately 10 times too
small to account for the observed increase in parallel elastic
stiffness of muscle fibres upon calcium activation [19].
3. TITIN CONTRIBUTES TO MUSCLE Force enhancement in muscle is an increase in steady-
PASSIVE TENSION state force after active stretch above the isometric force at
Numerous studies have described the passive force– a corresponding length [6], which has been attributed to
extension relationship for intact myofibrils, as well as for recruitment of a passive element [33]. In addition, the
individual titin molecules or portions thereof [14,20,28]. passive force after deactivation of an actively stretched
At shorter sarcomere lengths, passive stretch straightens muscle fibre is higher than the force produced after pas-
the folded Ig domains of I-band titin with little increase sive stretch, or after deactivation from an isometric
in passive tension [28,29]. At longer sarcomere lengths, contraction at a corresponding length [18], further
the PEVK domain elongates and the passive tension suggesting that ‘passive force enhancement’ is owing to
increases steeply [26,30]. PEVK behaves as an entropic recruitment of a passive element, namely titin [18].

Proc. R. Soc. B (2012)

 Is titin a winding filament? K. C. Nishikawa et al. 983

Joumaa et al. [34] measured passive force enhancement in shorter lengths compared with resting muscle; the titin
myofibrils in which active force production was prevented spring is approximately 10 per cent shorter and approxi-
by removal of troponin C. Like Labeit et al. [20], they also mately 2.5 times stiffer in active soleus muscles than in
observed a Ca2þ-induced increase in titin-based stiffness, resting soleus muscles. By contrast, no change in the
but the increase was too small to account for passive force length or stiffness of the titin spring is observed upon acti-
enhancement. They proposed that passive force enhance- vation of muscles from mdm mice, which are missing
ment not only requires Ca2þ influx, but also requires numerous amino acids in the N2A region [39]. These
cross-bridge formation or active force production [34]. observations are consistent with the hypothesis that bind-
In myofibrils stretched beyond overlap of the thick and ing of N2A titin to thin filaments on Ca2þ influx shortens
thin filaments (sarcomere length (SL) . 3.8 mm), Leo- and stiffens the titin spring.
nard & Herzog [35] also found evidence for both an
activation-dependent and a force-dependent increase in
titin stiffness. These data demonstrate that titin stiffness 6. Ca21-DEPENDENT N2A –ACTIN INTERACTIONS
is increased by Ca2þ influx and force development in active CONTRIBUTE TO LENGTH –TENSION
muscle. The winding filament hypothesis, described RELATIONSHIP
below, provides molecular mechanisms for effects of If N2A titin could bind at multiple locations along the
both Ca2þ influx and cross-bridge cycling on titin-based length of the thin filaments, then muscle force and vel-
tension in active sarcomeres. ocity [41] would remain relatively constant as sarcomere
length increased (figure 2a,b). In this way, Ca2þ-depen-
dent binding of N2A titin to thin filaments would
5. Ca21-DEPENDENT BINDING OF TITIN TO reinforce the plateau in the active length –tension relation-
THIN FILAMENTS ship, which corresponds to the cross-bridge free zone in
The N2A region (figure 1b,c) is in the ideal position for the middle of the thick filaments [42].
modulation of titin stiffness through Ca2þ-dependent An unsolved question in muscle physiology is why car-
binding to thin filaments. Binding of titin to actin at diac muscles, in contrast to skeletal muscles, have no
this location would eliminate low-force straightening of plateau in their active length – tension relationship. The
proximal tandem Ig domains in the I-band that normally plateau is expected to be at least as broad or broader in
occurs upon passive stretch of myofibrils at slack length cardiac than in skeletal muscles [42] on the basis of varia-
[14]. Furthermore, when Ca2þ-activated sarcomeres are bility in thin filament lengths [43]. In contrast to skeletal
stretched, the PEVK segment will elongate at high muscles, which express the N2A isoform of titin, cardiac
force. If Ca2þ-dependent binding between N2A titin myocytes in the ventricles of mice and rats express only
and thin filaments could be prevented, then active force the N2B isoform [44], which shows no Ca2þ-dependent
production should decrease at short sarcomere lengths. binding to actin [45]. Therefore, like muscles from mdm
Any strain developed in titin would straighten the mice, cardiac myocytes are predicted to lack binding of
tandem Ig segments at low force rather than extend the N2A titin to the thin filaments. Cardiac myocytes of
PEVK segment at higher force. Thus, any contribution rats and mice exhibit no plateau in active force [46,47].
of titin to the total active force would be reduced. By contrast, cardiac myocytes from trout express the
The available evidence suggests that titin binds to thick larger N2BA isoform, which includes both N2A and
filaments in the presence of Ca2þ. However, the location N2B isoforms, and is therefore predicted to exhibit
of the binding site within the giant titin molecule remains Ca2þ-dependent binding to thin filaments. The increased
unknown. In skeletal muscle, a product of titin proteolysis compliance of trout myocardium provided by the N2AB
(T2) binds to actin and reconstituted thin filaments with isoform allows for greater extension during diastolic fill-
high affinity in a Ca2þ-dependent manner [36]. This T2 ing. As predicted by the hypothesis, trout cardiac
fragment (approx. 2 MDa, approx. 920 nm) includes myocytes, like skeletal muscles, exhibit a plateau in the
the C-terminal and A-band FNIII and Ig domains, length – tension relationship [48].
PEVK and part of the N2A region [37]. The N2A An important difference between cardiac and skeletal
region contains an epitope that binds p94/calpain3 [38]. muscle is that cardiac muscle is never stretched while
It is thought that the T2 fragment forms when p94/cal- active, although it does experience passive lengthening
pain3 digests titin near this binding site in the N2A during diastolic filling [49]. Thus, there is no need for a
region. In the absence of Ca2þ, there is a weak interaction Ca2þ-dependent increase in titin stiffness in cardiac muscle.
between T2 and thin filaments, but when Ca2þ increases By contrast, skeletal muscle is routinely active during stretch
(pCa ¼ 4.5), the affinity of T2 for thin filaments increases [49–51] and possesses an isoform of titin (N2A) that
[36]. The location of the Ca2þ-dependent actin-binding is hypothesized to increase in stiffness on Ca2þ activation.
site within the large T2 fragment remains to be identified.
We suggest that this Ca2þ-dependent actin-binding
site is located at the N-terminal end of the T2 fragment 7. DOES CROSS-BRIDGE CYCLING ROTATE
in the N2A region of titin (figure 1b,c). Our recent work THE THIN FILAMENTS?
on intact soleus muscles of mutant mice (mdm) suggests In active muscle sarcomeres, cross-bridges probably
that an epitope of titin, in or near the N2A region, rotate as well as translate the thin filaments. Given the
binds to thin filaments in the presence of Ca2þ [39]. In structure of the thick and thin filaments, Morgan [10]
mice, the mdm mutant is characterized by a 779 base- showed that maintenance of stereospecific binding
pair deletion in the N2A region of the titin gene [40]. between an actin monomer and its three neighbouring
During rapid unloading in muscles of wild-type mice, thick filaments requires the thin filaments to rotate by
activation shifts the force– displacement curve towards approximately 288 as the myosin heads translate the

Proc. R. Soc. B (2012)

984 K. C. Nishikawa et al. Is titin a winding filament?

end of
(a) cross-bridge thin
overlap zone filament
2.4 µm

N2A
(b) 2.6 µm

(c)

(d)

Figure 2. Schematic illustrating the winding filament hypothesis. (a,b) Ca2þ-dependent binding of N2A to thin filaments con-
tributes to length–tension relationship. If N2A (red) binds non-selectively to thin filaments (blue) in the presence of Ca2þ, and
if the binding site depends on sarcomere length at the time of Ca2þ influx, then a plateau is predicted in active force at sarco-
mere lengths between (a) 2.4 and (b) 2.6 mm in rabbit psoas muscle. (c,d) Cross-bridge cycling results in titin winding.
(c) Cycling of the cross-bridges winds PEVK on the thin filaments (arrow indicates direction of rotation). In the model, the
winding angle depends only on sarcomere geometry. (d) Stretch of an active sarcomere extends the PEVK segment and
enhances the active force.

length of one actin monomer (approx. 5.5 nm). This rotation of thin filaments during force development and
would produce one full rotation of the thin filaments for active shortening.
every approximately 71.5 nm of translation. Changes in Z-disc structure upon muscle activation are
Rotation of actin filaments by heavy meromyosin has also consistent with thin filament rotation. In the Z-disc,
been observed in vitro [52 – 54]. Nishizaka et al. [53] each thin filament is anchored to its neighbours by four
observed that myosin heads produce a right-handed a-actinin ‘lanyards’ that form a small square pattern in
torque on actin filaments along their long axis, which resting sarcomeres when viewed in cross section [57].
winds up the right-handed twists of the actin double When muscles develop isometric force or shorten isotoni-
helix. In vitro, where the interactions between actin and cally, Z-disc structure changes from a small square to a
myosin are more diffuse than in muscle sarcomeres, basket-weave pattern [57]. This change in the orientation
actin filaments complete one full turn of rotation for of a-actinin is consistent with thin filament rotation [58].
every 1 mm of translation [54].
In a muscle sarcomere, because the actin filaments are
anchored to the Z-disc, cross-bridges could produce 8. THE WINDING FILAMENT HYPOTHESIS
twisting of the thin filaments in addition to rotation, Because titin is bound to thick filaments in the A-band
which could reduce the helical pitch of the actin helix and to thin filaments in the Z-disc [23], rotation of thin
[53]. Using X-ray diffraction, changes in the helical pitch filaments by the cross-bridges must lead inevitably to
of thin filaments have been observed in active muscle winding of titin upon them. Rotation of the thin filaments
fibres [55,56], although a confounding factor is that the by the cross-bridges would also produce a torque in
thin filaments also change in length during activation. a-actinin in the Z-disc. Winding of titin on the thin fila-
Bordas et al. [55] observed no difference in helical pitch ments is predicted to change the length and stiffness of
between fibres at rest and maximum isometric force, PEVK, storing elastic potential energy during isometric
although the thin filaments became longer at maximum iso- force development and active stretch. This energy could
metric force. The helical pitch of actin filaments decreased be recovered during active shortening.
during unloaded shortening. Tsaturyan et al. [56] found Unwinding of titin from the thin filaments could be
that thin filaments were more twisted in rigour than at prevented by electrostatic interactions between PEVK
rest. For a 1 mm long thin filament, the observed decrease and the thin filaments [31,45,59]. Spontaneous dis-
in helical pitch between rigour and rest corresponds to sociation rates of PEVK bound to actin are low, and the
a 2708 right-handed twist of the thin filaments. These force required to break the bonds is approximately equal
observations are consistent with the hypothesis that cross- to the force required to break an actomyosin cross-
bridge interactions with actin produce a right-handed bridge [59]. Unwinding of PEVK from the thin filaments

Proc. R. Soc. B (2012)

 Is titin a winding filament? K. C. Nishikawa et al. 985

bound PEVK
thin filament

attachment fre
eP
EV
K
thick filament + distal Ig

lateral view axial view

r φ
Xw (bound length) dXw
rdf q

x( dd1
fre
el
h eng
th)

h q x

d1 d2–d1
d2–d1
d2

Figure 3. Kinematics of titin winding. Winding angle (u) is the angle formed between the titin filament and a line (h) parallel to
the Z-disc. In the model, the winding angle is determined by sarcomere geometry and increases with sarcomere length. As the
winding angle (u) increases, the length of free PEVK (x) will decrease for a given angle of thin filament rotation ( f ). Abbrevi-
ations: d1, distance from the Z-disc to the point at which bound PEVK becomes free; d2, distance from the Z-disc to the distal
(C-terminal) end of PEVK; r, radius.

is hypothesized to occur (i) during active shortening [59] model, the winding angle is determined only by sarcomere
at low loads when the combined PEVK –actin and cross- geometry, and thus increases with sarcomere length. As thin
bridge forces are too low to hold the torques in titin and filament rotation (f ) increases, the length of the free PEVK
a-actinin, as well as (ii) during muscle relaxation. segment will decrease and the stress in this segment will
increase, thereby increasing its effective stiffness. However,
the edge between free and bound PEVK will also advance
9. TITIN WINDING DURING ISOMETRIC FORCE towards the M-line, reducing PEVK strain. In the model,
DEVELOPMENT AND ACTIVE STRETCH titin winding varies from approximately 2008 at 2.4 mm
During isometric force development, we propose that SL to approximately 308 at 3.7 mm SL. The proportion of
winding of titin on the thin filaments proceeds until the winding owing to twisting versus rotation of actin will
radial component of the cross-bridge force is equal to the depend on the rotational stiffness of actin versus a-actinin.
sum of the radial forces in titin and a-actinin. As force A nonlinear ordinary differential equation was used to
develops, the length of bound PEVK that is wound upon simulate the kinematics of PEVK winding and resulting
the thin filaments would increase, increasing strain, or stiff- axial forces for a given profile of thin filament rotation
ness, or both, in the free portion of PEVK (figure 2c). f(t) and sarcomere geometry. In the axial direction, the
When active sarcomeres are lengthened by application of total force is the sum of the axial forces produced by
an external force (figure 2d), the work done in elongating PEVK and the cross-bridges. In the axial plane, the
free PEVK would also be stored as elastic potential sum of the torques owing to radial forces produced by
energy, resulting in force enhancement at low energy cost. PEVK in the I-band and a-actinin in the Z-disc is equal
We developed a kinematic model (figure 3) to quantify and opposite to the torque produced by the cross-bridge
the effects of thin filament rotation on PEVK during iso- forces. Cross-bridge forces were calculated based on
metric force development and active stretch. The model is Pavlov et al. [60], PEVK forces were calculated based
based on a sarcomere structure similar to rabbit psoas on Linke et al. [14] and the torque in a-actinin was
muscle [14,60], with a thin filament diameter of 10 nm modelled as an exponential spring.
[61] and a titin filament diameter of 4 nm [23,62]. The Using this model, we simulated force enhancement on
length of the PEVK segment is approximately 104 nm the descending limb of the force–length relationship by
at SL ¼ 2.6 mm [14]. calculating the axial forces produced by the cross-bridges
Increasing strain and stiffness of PEVK owing to thin and titin in sarcomeres activated at different initial lengths,
filament rotation depend on the winding angle upon the and then stretched while active (figure 4). The results are
thin filament (figure 3). The winding angle (u) is defined qualitatively similar to experimental observations [63].
as the angle formed between the titin filament and a line These results demonstrate that the winding filament
(h) parallel to the Z-disc at the point at which PEVK is no hypothesis accounts for the observed pattern of residual
longer in contact with the thin filament (figure 3). In the force enhancement in actively stretched muscle.

Proc. R. Soc. B (2012)

986 K. C. Nishikawa et al. Is titin a winding filament?

0.16

0.14

0.12
stress (N mm–2)
0.10

0.08

0.06

0.04

0.02

0
2400 2600 2800 3000 3200 3400 3600 3800 4000
sarcomere length (nm)
Figure 4. Simulation of residual force enhancement on the descending limb of the force– length relationship. Predicted axial
stress owing to cross-bridges (green) and PEVK (red). Total axial stress (blue) is the sum of axial stress owing to cross-bridges
and PEVK. Baselines show steady-state isometric stress. Branches show increased stress owing to stretch. Residual force
enhancement (black) is the increase in force owing to active stretching above the isometric force at the corresponding length.

10. TITIN WINDING DURING ACTIVE SHORTENING nonlinearity in shortening rate during after-loaded
During active shortening, cross-bridge interactions trans- isotonic contractions [66].
late the thin filaments towards the M-line, decreasing Hill [66] initially claimed that the heat of shortening
elastic energy storage in PEVK, but also rotate the thin increases monotonically with shortening velocity in active
filaments, increasing energy storage. Owing to the depen- muscles. His measurements were later revised, however,
dence of cross-bridge force, duty factor and step size on to show that the shortening heat levels off at about
shortening velocity [64,65], PEVK would increasingly 0.5Vmax and decreases thereafter despite the increase in
unwind from the thin filaments as the cross-bridge force shortening velocity [67]. Although the nature of this
declines with shortening velocity. In fact, without such relationship was well predicted by Huxley’s [68] two-state
unwinding, the amplitude of muscle shortening will be attachment model, the model does not specify the source
limited unrealistically by the bound PEVK. of the energy needed to increase contraction velocity
These considerations lead to the prediction that net above 0.5Vmax. (Note that this energy cannot come from
storage or recovery of elastic potential energy during ATP hydrolysis because the heat of shortening is declin-
active shortening should depend on the shortening vel- ing.) The winding filament model explains the levelling
ocity. For example, when a muscle shortens slowly and subsequent decline in ATPase rate with shortening vel-
against a load that is close to its maximum isometric ocity as an increase in the rate of conversion of elastic
force, the recovery of elastic energy from PEVK owing energy stored in PEVK to kinetic energy.
to thin filament translation will be small relative to the
energy stored in PEVK owing to thin filament rotation.
Furthermore, the high cross-bridge force and duty 11. INSTANTANEOUS ELASTICITY OF
factor will tend to prevent PEVK unwinding. Thus, the ACTIVE SARCOMERES
muscle will exhibit a net storage of elastic energy in It is widely accepted that the instantaneous elasticity of
PEVK despite shortening. active muscle fibres resides solely in the cross-bridges
By contrast, when a muscle shortens against a small [69]. Supporting evidence includes the observations that
load at a velocity close to Vmax, the cross-bridge force the change in length required to reduce the transient
and duty factor will be smaller and the step size will be force to zero (4–14 nm per half-sarcomere [70]) is of the
larger, so the rate of elastic energy recovery from PEVK order of a single cross-bridge step, and that the elastic
owing to thin filament translation would exceed the rate behaviour of stretched muscle fibres varies in direct
of energy storage owing to thin filament rotation. In proportion to the overlap between the thick and thin
addition, the reduced cross-bridge force would permit filaments [69].
PEVK unwinding, which would further increase recovery However, there is some disagreement about whether
of elastic potential energy. the cross-bridges alone can account for the instantaneous
Likewise, the winding filament model predicts that elasticity of active sarcomeres [71]. Working with permeabi-
shortening velocity should decline over time during iso- lized, isolated muscle fibres, Galler & Hilber [72] reported
tonic contractions at small loads as the elastic energy that the length change required to reduce the transient force
stored in PEVK is dissipated. Shortening velocity should to zero increased from 6 to 18 nm per half-sarcomere when
decrease faster at smaller loads, because stored PEVK the temperature was raised from 6 to 348C. They concluded
energy would be recovered at a faster rate. In this way, that, at higher temperatures, the length change required to
the winding filament model accounts for the observed reduce the active force to zero was too great to be explained

Proc. R. Soc. B (2012)

 Is titin a winding filament? K. C. Nishikawa et al. 987

by cross-bridge compliance alone. In isolated fibres of (a) History dependence

horseshoe crab telson, which exhibit extremely long sarco- History-dependent changes in active force production
mere lengths (approx. 7 mm), the length change required include enhancement of force with stretch and depression
to reduce the force to zero was 210 nm per half-sarcomere of force with shortening [6,63,77]. The steady-state force
[73]. This led Sugi et al. [73] to suggest that titin might produced by muscles after active shortening is less than
contribute to instantaneous elasticity. the isometric force at a corresponding length, and likewise
The winding filament model predicts that PEVK strain the steady-state force following active lengthening is higher
owing to winding will contribute to the instantaneous than the isometric force at a corresponding length. These
elasticity of active muscle. In the winding filament history-dependent properties of active muscle are exactly
model, PEVK strain (i) increases nonlinearly with thin those expected of springs, which produce greater tensile
filament rotation, (ii) is greatest on the plateau of the force when stretched and less tensile force when shortened,
length – tension relationship, and (iii) decreases rapidly in proportion to their change in length [78].
with increasing sarcomere length. The model predicts The winding filament model provides a mechanism for
that PEVK strain will accumulate at a rate of approxi- both depression of force with shortening and enhancement
mately 30 nm per full rotation of the thin filaments at of force with stretch. During active shortening, energy
optimal length (2.4 mm in rabbit psoas); PEVK strain stored in PEVK will be converted to kinetic energy, and
will accumulate more slowly on the descending limb of muscle force will decrease in direct proportion to the dis-
the length – tension relationship. tance shortened. Owing to velocity-dependent unwinding
The non-uniformity in sarcomere length that develops of PEVK from the thin filaments, force depression will
during isometric force development may also contribute also increase with shortening velocity. After shortening, a
to the instantaneous elasticity of single muscle fibres muscle will recover force as the cycling cross-bridges re-
upon quick release [73]. As muscle fibres contract iso- wind PEVK upon the thin filaments. During active stretch,
metrically, imbalances in the forces produced in adjacent the work done in stretching will extend PEVK, storing elas-
half-sarcomeres lead to stretching of the weaker half- tic strain energy. This added force will increase with the
sarcomeres by the stronger ones, displacing their A-bands distance stretched.
until stretching of their titin equalizes the forces. In this The sliding filament theory predicts that the total
way, titin molecules stretched during isometric force devel- active force on the descending limb of the force– length
opment could contribute to the instantaneous elasticity curve should never exceed the maximum isometric force
observed upon quick release. at optimum overlap. The active steady-state force follow-
Sugi et al. [73] worked with single muscle fibres from ing muscle stretch on the descending limb of the force–
horseshoe crab telson, which are unusual in possessing length curve in fact can exceed the maximum isometric
long (7 mm) sarcomeres that lack the distinct M-line force [18]. Unlike the sliding filament theory, the winding
structures that are typically found in sarcomeres of filament model predicts that active force can rise above
vertebrate muscle. However, the development of half-sarco- the maximum isometric force on the descending limb of
mere non-uniformity during isometric force development the force– length curve.
appears to be a feature of vertebrate sarcomeres as well
[60,74]. In rabbit psoas muscle, A-band displacements of (b) Low cost of lengthening contractions
approximately 60 nm were observed at optimum length During active stretch, muscles require much less energy to
and maximum isometric force. The observed A-band dis- produce an equivalent force than when they contract con-
placement is also expected to contribute to instantaneous centrically [7]. It appears as though some of the work
elasticity. done in stretching an active muscle can be absorbed to
During the past decade, the view that single cross- enhance force. In the winding filament model, when an
bridges represent the independent force-generating units external force stretches a sarcomere, the work done in
of muscle has been modified, in light of thick and thin stretching PEVK is stored as elastic energy, which increases
filament compliance, to include the thick and thin fila- muscle force and can be recovered during shortening.
ment lattice as a whole [75]. In the winding filament
model, the unit of force generation also includes the elas-
tic titin and a-actinin filaments because they transmit 13. CONCLUSION
radial forces produced during cross-bridge interactions. The winding filament model provides a simple mechanism
This view is supported by the observations of Shimamoto by which the PEVK segment of titin contributes to muscle
et al. [76] that treatment of single myofibrils with antibodies force development and active shortening. It is consistent
to a-actinin substantially reduces force production as well with structural and viscoelastic properties of muscle
as sarcomere length non-uniformity. sarcomeres in general, and of titin in particular. It builds
on the swinging cross-bridge–sliding filament theory
and increases its explanatory power. In the winding fila-
12. EXPLANATORY VALUE OF THE WINDING ment model, Ca2þ-dependent binding of N2A to the thin
FILAMENT MODEL filaments prevents low-force straightening of proximal
The winding filament model is not intended to replace the tandem Ig domains in the I-band that occurs upon passive
sliding filament theory. Instead, by adding an elastic stretch of skeletal myofibrils at slack length. In this way,
element (i.e. titin) inside active muscle sarcomeres that it contributes to the plateau in force and velocity at
is modulated by Ca2þ influx and cross-bridge cycling, intermediate sarcomere lengths in skeletal muscle.
the winding filament model accounts for a wide range of In the winding filament model, the cross-bridges rotate
well-established phenomena that remain problematic the thin filament with each cycle of ATP hydrolysis, wind-
under the sliding filament theory alone. ing PEVK upon the thin filaments. This winding stores

Proc. R. Soc. B (2012)

988 K. C. Nishikawa et al. Is titin a winding filament?

elastic energy in PEVK during isometric force develop- 10 Morgan, R. S. 1977 Actin rotates as myosin translates.
ment. The stored energy is recovered during shortening J. Theor. Biol. 76, 769 –771. (doi:10.1016/0022-5193
at rates that depend on the external load. Interactions (77)90261-2)
between the cycling cross-bridges and PEVK regulate the 11 Maruyama, K. 1976 Connectin, an elastic protein from
rate of conversion of elastic potential energy to kinetic myofibrils. J. Biochem. 80, 405– 407.
12 Wang, K., McClure, J. & Tu, A. 1979 Titin: major myo-
energy, endowing active muscle with intrinsic mechanical
fibrillar components of striated muscle. Proc. Natl Acad.
stabilization to load perturbations. During active lengthen- Sci. USA 76, 3698–3702. (doi:10.1073/pnas.76.8.3698)
ing, external work done on PEVK increases muscle force, 13 Horowitz, R. & Podolsky, R. J. 1987 The positional stab-
reducing the energetic cost of force production. ility of thick filaments in activated skeletal muscle
A definitive test of the winding filament hypothesis depends on sarcomere length: evidence for the role of
awaits new developments in nanoscale imaging that titin filaments. J. Cell Biol. 105, 2217–2223. (doi:10.
would enable visualization of the movements of approxi- 1083/jcb.105.5.2217)
mately 2 nm diameter fibres. Although no direct 14 Linke, W. A., Ivemeyer, M., Mundel, P., Stockmeier,
evidence supports the idea that the cross-bridges wind M. R. & Kolmerer, B. 1998 Nature of PEVK –titin elas-
titin upon the thin filaments, the explanatory value of ticity in skeletal muscle. Proc. Natl Acad. Sci. USA 95,
8052–8057. (doi:10.1073/pnas.95.14.8052)
the idea is evident. Although some details remain to be
15 Gregorio, C. C., Granzier, H., Sorimachi, H. & Labeit, S.
quantified, the hypothesis provides numerous testable 1999 Muscle assembly: a titanic achievement. Curr.
predictions that will encourage new directions for Opin. Cell Biol. 11, 18–25. (doi:10.1016/S0955-0674
research on the mechanisms of muscle contraction. (99)80003-9)
P. Aerts, M. Azizi, T. Daniel, M. Dickinson, 16 Krüger, M. & Linke, W. A. 2011 The giant protein titin:
S. Hempleman, W. Herzog, K. Hollander, J. Van Leeuwen, a regulatory node that integrates myocyte signalling path-
F. Nelson, D. Pierotti, J. Pilarski, A. Ruina, T. Roberts, ways. J. Biol. Chem. 286, 9905– 9912. (doi:10.1074/jbc.
P. Service, T. Sugar and S. Warburton contributed to the R110.173260)
development of the ideas presented here. P. Aerts, F. Nelson, 17 Reich, T. E., Lindstedt, S. L., LaStayo, P. C. & Pierotti,
T. Roberts and J. Van Leeuwen provided suggestions for D. J. 2000 Is the spring quality of muscle plastic? Am
improving the manuscript. A. K. Lappin, J. Pilarski and J. Physiol. 278, R1661–R1666.
E. Zepnewski collected experimental data that motivated the 18 Herzog, W. & Leonard, T. R. 2002 Force enhancement
development of this theory. This research was supported by following stretching of skeletal muscle: a new mechanism.
grants IOS-0623791, IOS-0732949, IIS-0827688 and IOS- J. Exp. Biol. 205, 1275–1283.
1025806 from the National Science Foundation, and by 19 Campbell, K. S. & Moss, R. L. 2002 History-dependent
TRIF Growing Biotechnology grants from Northern Arizona mechanical properties of permeabilized rat soleus muscle
University. Research of D.K.P. and S.H.Y. was also fibers. Biophys. J. 82, 929– 943. (doi:10.1016/S0006-
supported by NSERC, CIHR, PWIAS and the Canada 3495(02)75454-4)
Research Chairs Programme. 20 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
REFERENCES the giant protein titin. Proc. Natl Acad. Sci. USA 100,
1 Huxley, A. F. & Niedergerke, R. 1954 Structural changes 13 716– 13 721. (doi:10.1073/pnas.2235652100)
in muscle during contraction. Nature 173, 971 –973. 21 Warren, C. M., Krzesinski, P. R. & Greaser, M. L. 2003
(doi:10.1038/173971a0) Vertical agarose gel electrophoresis and electroblotting
2 Huxley, H. & Hanson, J. 1954 Changes in the cross-stria- of high-molecular-weight proteins. Electrophoresis 24,
tions of muscle during contraction and stretch and their 1695–1702. (doi:10.1002/elps.200305392)
structural interpretation. Nature 173, 973 –976. (doi:10. 22 Wang, K., Ramirez-Mitchell, R. & Palter, D. 1984 Titin
1038/173973a0) is an extraordinarily long, flexible, and slender myofibril-
3 Huxley, A. F. 2000 Mechanics and models of the myosin lar protein. Proc. Natl Acad. Sci. USA 81, 3685–3689.
motor. Phil. Trans. R. Soc. Lond. B 355, 433 –440. (doi:10.1073/pnas.81.12.3685)
(doi:10.1098/rstb.2000.0584) 23 Funatsu, T., Kono, E., Higuchi, H., Kimura, S.,
4 Huxley, H. E. 2000 Past, present and future experiments Ishiwata, S., Yoshioka, T., Maruyama, K. & Tsukita, S.
on muscle. Phil. Trans. R. Soc. Lond. B 355, 539 –543. 1993 Elastic filaments in situ in cardiac muscle: deep-
(doi:10.1098/rstb.2000.0595) etch replica analysis in combination with selective
5 Herzog, W., Leonard, T. R., Joumaa, V. & Mehta, A. removal of actin and myosin filaments. J. Cell Biol. 120,
2008 Mysteries of muscle contraction. J. Appl. Biomech. 711 –724. (doi:10.1083/jcb.120.3.711)
24, 1 –13. 24 Bennett, P. M., Hodkin, T. E. & Hawkins, C. 1997
6 Abbott, B. C. & Aubert, X. M. 1952 The force exerted Evidence that the tandem Ig domains near the end of
by active striated muscle during and after change of the muscle thick filament form an inelastic part of the
length. J. Physiol. 117, 77–86. I-band titin. J. Struct. Biol. 120, 93– 104. (doi:10.1006/
7 Bigland-Ritchie, B. & Woods, J. J. 1976 Integrated elec- jsbi.1997.3898)
tromyogram and oxygen uptake during positive and 25 Muhle-Goll, C., Habeck, M., Cazorla, O., Nilges, M.,
negative work. J. Physiol. (Lond.) 260, 267– 277. Labeit, S. & Granzier, H. 2001 Structural and functional
8 Kushmerick, M. J. & Davies, R. E. 1969 The chemical studies of titin’s fn3 modules reveal conserved surface
energetics of muscle contraction. II. The chemistry, effi- patterns and binding to myosin S1: a possible role in
ciency and power of maximally working sartorius muscle. the Frank –Starling mechanism of the heart. J. Mol.
Proc. R. Soc. Lond. B 174, 315 –353. (doi:10.1098/rspb. Biol. 313, 431 –477. (doi:10.1006/jmbi.2001.5017)
1969.0096) 26 Gautel, M. & Goulding, G. 1996 A molecular map of
9 Julian, F. J. & Morgan, D. L. 1979 The effects of tension titin/connectin elasticity reveals two different mechan-
on non-uniform distribution of length changes applied to isms acting in series. FEBS Lett. 385, 11– 14. (doi:10.
frog muscle fibres. J. Physiol. (Lond.) 293, 379– 392. 1016/0014-5793(96)00338-9)

Proc. R. Soc. B (2012)

 Is titin a winding filament? K. C. Nishikawa et al. 989

27 Linke, W. A., Ivemeyer, M., Labeit, S., Hinssen, H., 44 Cazorla, O., Freiburg, A., Helmes, M., Centner, T.,
Rüegg, J. C. & Gautel, M. 1997 Actin –titin interaction McNabb, M., Wu, Y., Trombitas, K., Labeit, S. &
in cardiac myofibrils: probing a physiological role. Granzier, H. 2000 Differential expression of cardiac
Biophys. J. 73, 905 –919. (doi:10.1016/S0006-3495(97) titin isoforms and modulation of cellular stiffness. Circ.
78123-2) Res. 86, 59–67.
28 Wang, K., McCarter, R., Wright, J., Beverly, J. & 45 Yamasaki, R. et al. 2001 Titin –actin interaction in mouse
Ramirez-Mitchell, R. 1991 Regulation of skeletal myocardium: passive tension modulation and its regu-
muscle stiffness and elasticity by titin isoforms: a test of lation by calcium/S100A1. Biophys. J. 81, 2297– 2313.
the segmental extension model of resting tension. Proc. (doi:10.1016/S0006-3495(01)75876-6)
Natl Acad. Sci. USA 88, 7101–7105. (doi:10.1073/ 46 Weiwad, W. K. K., Linke, W. A. & Wussling, M. H. P. 2000
pnas.88.16.7101) Sarcomere length–tension relationship of rat cardiac myo-
29 Granzier, H. L. & Labeit, S. 2004 The giant protein titin: cytes at lengths greater than optimum. J. Mol. Cell. Cardiol.
a major player in myocardial mechanics, signaling and 32, 247–259. (doi:10.1006/jmcc.1999.1069)
disease. Circ. Res. 94, 284 –295. (doi:10.1161/01.RES. 47 Shiels, H. A. & White, E. 2008 The Frank–Starling
0000117769.88862.F8) mechanism in vertebrate cardiac myocytes. J. Exp. Biol.
30 Trombitas, K., Greaser, M., French, G. & Granzier, H. 211, 2005– 2013. (doi:10.1242/jeb.003145)
1998 PEVK extension of human soleus muscle titin 48 Patrick, S. M., Hoskins, A. C., Kentish, J. C., White, E.,
revealed by immunolabeling with the anti-titin antibody Shiels, H. A. & Cazorla, O. 2010 Enhanced length-
9D10. J. Struct. Biol. 122, 188– 196. (doi:10.1006/jsbi. dependent Ca2þ activation in fish cardiomyocytes per-
1998.3984) mits a large operating range of sarcomere lengths.
31 Linke, W. A., Kulke, M., Li, H., Fujita-Becker, S., Neagoe, J. Mol. Cell. Cardiol. 48, 917–924. (doi:10.1016/j.
C., Manstein, D. J., Gautel, M. & Fernandez, J. M. 2002 yjmcc.2010.02.008)
PEVK domain of titin: an entropic spring with actin bind- 49 King, N. M., Mathawasin, M., Nedrud, J., Harrell, N.,
ing properties. J. Struct. Biol. 137, 194–205. (doi:10.1006/ Chung, C. S., Helmes, M. & Granzier, H. 2010 Mouse
jsbi.2002.4468) intact cardiac myocyte mechanics: cross-bridge and
32 Magid, A. & Law, D. J. 1985 Myofibrils bear most of titin-based stress in unactivated cells. J. Gen. Physiol.
the resting tension in frog skeletal muscle. Science 230, 137, 81– 91. (doi:10.1085/jgp.201010499)
1280 –1282. (doi:10.1126/science.4071053) 50 Biewener, A. & Roberts, T. J. 2000 Muscle and tendon
33 Edman, K. A. P. & Tsuchiya, T. 1996 Strain of passive contributions to force, work and elastic energy savings:
elements during force enhancement by stretch in frog a comparative perspective. Exerc. Sports Sci. Rev. 28,
muscle fibres. J. Physiol. 490, 191 –205. 99– 107.
34 Joumaa, V., Leonard, T. & Herzog, W. 2008 Residual 51 Dickinson, M. H., Farley, C. T., Full, R. J., Koehl,
force enhancement in myofibrils and sarcomeres. M. A. R., Kram, R. & Lehman, S. 2000 How animals
Proc. R. Soc. B 275, 1411–1419. (doi:10.1098/rspb. move: an integrative view. Science 288, 100 –106.
2008.0142) (doi:10.1126/science.288.5463.100)
35 Leonard, T. R. & Herzog, W. 2010 Regulation of muscle 52 Tanaka, Y., Ishijima, A. & Ishiwata, S. 1992 Super helix
force in the absence of actin –myosin based cross-bridge formation of actin filaments in an in vitro motile system.
interaction. Am. J. Physiol. Cell Physiol. 299, 14–20. Biochim. Biophys. Acta 159, 94–98. (doi:10.1016/0167-
(doi:10.1152/ajpcell.00049.2010) 4838(92)90079-S)
36 Kellermayer, M. S. Z. & Granzier, H. L. 1996 Calcium- 53 Nishizaka, T., Yagi, T., Tanaka, Y. & Ishiwata, S. 1993
dependent inhibition of in vitro thin-filament motility by Right-handed rotation of an actin filament in an in vitro
native titin. FEBS Lett. 380, 281 –286. (doi:10.1016/ motile system. Nature 361, 269–271. (doi:10.1038/
0014-5793(96)00055-5) 361269a0)
37 Suzuki, J., Kimura, S. & Maruyama, K. 1994 Electron 54 Sase, I., Miyata, H., Ishiwata, S. & Kinosita, K. 1997
microscopic filament lengths of connectin and its frag- Axial rotation of sliding actin filaments revealed by
ments. J. Biochem. (Tokyo) 116, 406 –410. single-fluorophore imaging. Proc. Natl Acad. Sci. USA
38 Hayashi, C. et al. 2008 Multiple molecular interactions 94, 5646–5650. (doi:10.1073/pnas.94.11.5646)
implicate the connectin/titin N2A region as a modulating 55 Bordas, J., Svensson, A., Rothery, M., Lowy, J., Diakun,
scaffold for p94/calpain 3 activity in skeletal muscle. G. P. & Boesecke, P. 1999 Extensibility and symmetry of
J. Biol. Chem. 283, 14 801– 14 814. actin filaments in contracting muscles. Biophys. J. 77,
39 Monroy, J. A., Powers, K. L., Gilmore, L. A., Uyeno, 3197– 3207. (doi:10.1016/S0006-3495(99)77150-X)
T. A. & Nishikawa, K. C. 2011 Ca2þ-activation of skel- 56 Tsaturyan, A. K., Koubassova, N., Ferenczi, M. A.,
etal muscle: not just the thin filament? Integr. Comp. Narayanan, T., Roessle, M. & Bershitsky, S. Y. 2005
Biol. 51, e94. (doi:10.1093/icb/icr008) Strong binding of myosin heads stretches and twists
40 Garvey, S. M., Rajan, C., Lerner, A. P., Frankel, W. N. & the actin helix. Biophys. J. 88, 1902–1910. (doi:10.
Cox, G. A. 2002 The muscular dystrophy with myositis 1529/biophysj.104.050047)
(mdm) mouse mutation disrupts skeletal muscle-specific 57 Goldstein, M. A., Schroeter, J. P. & Sass, R. L. 1990
domain of titin. Genom 79, 146 –149. (doi:10.1006/ Two structural states of the vertebrate Z band. Electron
geno.2002.6685) Microsc. Rev. 3, 227–248. (doi:10.1016/0892-0354(90)
41 Edman, K. A. P. 1979 The velocity of unloaded shorten- 90003-B)
ing and its relation to sarcomere length and isometric 58 Jarosch, R. 2000 Muscle force arises by actin filament rota-
force in vertebrate muscle fibers. J. Physiol. 291, tion and torque in the Z-filaments. Biochem. Biophys. Res.
143 –159. Commun. 270, 677–682. (doi:10.1006/bbrc.1999.1971)
42 Allen, D. G. & Kentish, J. C. 1985 The cellular basis of 59 Bianco, P., Nagy, A., Kengyel, A., Szatmari, D., Marton-
the length –tension relation in cardiac muscle. J. Mol. falvi, Z., Huber, T. & Kellermayer, M. S. Z. 2007
Cell. Cardiol. 17, 821– 840. (doi:10.1016/S0022-2828 Interaction forces between F-actin and titin PEVK
(85)80097-3) domain measured with optical tweezers. Biophys. J. 93,
43 Robinson, T. F. & Winegrad, S. 1979 The measurement 2102– 2109. (doi:10.1529/biophysj.107.106153)
and dynamic implications of thin filament lengths in 60 Pavlov, I., Novinger, R. & Rassier, D. 2009 The
heart muscle. J. Physiol. 286, 607–619. mechanical behavior of individual sarcomeres of

Proc. R. Soc. B (2012)

990 K. C. Nishikawa et al. Is titin a winding filament?

myofibrils isolated from rabbit psoas muscle. stimulated frog muscle fibers near slack length.
Am. J. Physiol. Cell Physiol 297, C1211– C1219. J. Physiol. 269, 441–515.
(doi:10.1152/ajpcell.00233.2009) 71 Lappin, A. K., Monroy, J. A., Pilarski, J. Q., Zepnewski,
61 Shi, D., Somlyo, A. V., Somlyo, A. P. & Shao, Z. 2001 E. D., Pierotti, D. J. & Nishikawa, K. C. 2006 Storage
Visualizing filamentous actin on lipid bilayers by atomic and recovery of elastic potential energy powers ballistic
force microscopy in solution. J. Microsc. 201, 377 –382. prey capture in toads. J. Exp. Biol. 209, 2535–2553.
(doi:10.1046/j.1365-2818.2001.00844.x) (doi:10.1242/jeb.02276)
62 Higuchi, H., Nakauchi, Y., Maruyama, K. & Fujime, S. 72 Galler, S. & Hilber, K. 1998 Tension/stiffness ratio of
1993 Characterization of beta-connectin (titin 2) skinned rat skeletal muscle fibre types at various tempera-
from striated muscle by dynamic light scattering. tures. Acta Physiol. Scand. 162, 119 –126. (doi:10.1046/j.
Biophys. J. 65, 1906–1915. (doi:10.1016/S0006-3495 1365-201X.1998.0272f.x)
(93)81261-X) 73 Sugi, H., Akimoto, T., Kobayashi, T., Suzuki, S. &
63 Edman, K. A. P., Elzinga, G. & Noble, M. I. M. 1982 Shimada, S. 2000 Possible contribution of titin filaments
Residual force enhancement after stretch of contracting to the compliant series elastic component in horseshoe
frog single muscle fibers. J. Gen. Physiol. 80, 769 –784. crab skeletal muscle fibers. Adv. Exp. Med. Biol. 481,
(doi:10.1085/jgp.80.5.769) 371 –380. (doi:10.1007/978-1-4615-4267-4_22)
64 Finer, J. T., Mehta, A. D. & Spudich, J. A. 1995 74 Telley, I. A., Stehle, R., Ranatunga, K. W., Pfitzer, G.,
Characterization of single actin –myosin interactions. Stüssi, E. & Denoth, J. 2006 Dynamic behaviour of
Biophys. J. 68, S291– S296. (doi:10.1016/S0006-3495 half-sarcomeres during and after stretch in activated
(95)80187-6) rabbit psoas myofibrils: sarcomere asymmetry but no
65 Reconditi, M. et al. 2004 The myosin motor in muscle ‘sarcomere popping’. J. Physiol. 573, 173– 185. (doi:10.
generates a smaller and slower working stroke at higher 1113/jphysiol.2006.105809)
load. Nature 428, 578 –581. (doi:10.1038/nature02380) 75 Daniel, T. L., Trimble, A. C. & Chase, P. B. 1998
66 Hill, A. V. 1938 The heat of shortening and the dynamic Compliant realignment of binding sites in muscle: transi-
constants of muscle. Proc. R. Soc. Lond. B 126, 136 –195. ent behavior and mechanical tuning. Biophys. J. 74,
(doi:10.1098/rspb.1938.0050) 1611–1621. (doi:10.1016/S0006-3495(98)77875-0)
67 Hill, A. V. 1964 The effect of load on the heat of short- 76 Shimamoto, Y., Suzuki, M., Mikhailenko, S. V., Yasuda,
ening of muscle. Proc. R. Soc. Lond. B 159, 297 –318. K. & Ishiwata, S. 2009 Inter-sarcomere coordination in
(doi:10.1098/rspb.1964.0004) muscle revealed though individual sarcomere response
68 Huxley, A. F. 1973 A note suggesting that the cross- to quick stretch. Proc. Natl Acad. Sci. USA 106,
bridge attachment during muscle contraction may take 11 954– 11 959. (doi:10.1073/pnas.0813288106)
place in two stages. Proc. R. Soc. Lond. B 183, 83– 86. 77 Edman, K. A. 1975 Mechanical deactivation induced by
(doi:10.1098/rspb.1973.0006) active shortening in isolated muscle fibers of the frog.
69 Huxley, A. F. & Simmons, R. M. 1971 Proposed mech- J. Physiol. 246, 255–275.
anism of force generation in striated muscle. Nature 78 Monroy, J. A., Lappin, A. K. & Nishikawa, K. C. 2007
233, 533 –538. (doi:10.1038/233533a0) Elastic properties of active muscle: on the rebound?
70 Ford, L. E., Huxley, A. F. & Simmons, R. M. 1977 Exerc. Sports Sci. Rev. 35, 174 –179. (doi:10.1097/jes.
Tension responses to sudden length change in 0b013e318156e0e6)

Proc. R. Soc. B (2012)