Crossbridge Mechanism(s) Examined by Temperature Perturbation Studies on Muscle

Abstract

An overall view of the contractile process that has emerged from -temperature-studies on active muscle is outlined. In isometric muscle, a small rapid temperature-jump (T-jump) enhances an early, pre-phosphate release, step in the acto-myosin (crossbridge) ATPase cycle and induces a characteristic rise in force indicating that crossbridge force generation is endothermic (force rises when heat is absorbed). Sigmoidal temperature dependence of steady force is largely due to the endothermic nature of force generation. During shortening, when muscle force is decreased, the T-jump force generation is enhanced; conversely, when a muscle is lengthening and its force increased, the T-jump force generation is inhibited. Taking T-jump force generation as a signature of the crossbridge - ATPase cycle, the results suggest that during lengthening the ATPase cycle is truncated before endothermic force generation, whereas during shortening this step and the ATPase cycle, are accelerated; this readily provides a molecular basis for the Fenn effect.

Full text

Crossbridge Mechanism(s) Examined By
Temperature Perturbation Studies on Muscle
K.W. Ranatunga & M.E. Coupland
Muscle Contraction Group, Department of Physiology & Pharmacology, School of Medical
Sciences, University of Bristol, Bristol BS8 1TD, UK (e-mail: k.w.ranatunga@bristol.ac.uk)
1. Abstract
An overall view of the contractile process that has emerged from temperature-
studies on active muscle is outlined. In isometric muscle, asmall rapid tempera-
ture-jump (T-jump) enhances an early, pre-phosphate release, step in the acto-
myosin (crossbridge) ATPase cycle and induces a characteristic rise in for ce indi-
cating that crossbridge force generation is endothermic (force rises when heat is
absorbed).Sigmoidal temperature dependence of steady force is largely due to the
endothermic nature of force generation. During shortening, when muscle force is
decreased, the T-jump force generation is enhanced; conversely, when a muscle is
lengthening and its force increased, the T-jump force generation is inhibited. Tak-
ing T-jump force generation as a signature of the crossbridge - ATPase cycle, the
results suggest that during lengthening the ATPase cycle is truncated before endo-
thermic force generation, whereas during shortening this step and the ATPase cy-
cle, are accelerated; this readily provides a molecular basis for the Fenn effect.
2. Introduction
The process that underlies muscle contraction is an ATP-driven, cyclic inter-
action of crossbridges (myosin heads) between thick (M=myosin) and thin
(A=actin) filaments in a sarcomere (Huxley, 1957; Huxley, 1969); during a cycle,
a crossbridge (myosin head) attaches to actin, undergoes a conformational change
generating muscle force, and power, and then detaches. It has been known for over
half-century (Hadju, 1951) that muscle force is sensitive to temperature; in mam-
malian muscle, maximal active force increases by ~2-fold (Ranatunga & Wylie,
1983), shortening velocity increases by ~6-fold (Ranatunga, 1984) and power in-
creasesby >10-fold (Ranatunga, 1998) in warming fr om 10 °C to more physio-
logical temperatures (>30 °C). Thus, temperature-sensitivity determination and
temperature perturbation (T-jump) are obvious techniques to examine the mecha-
nisms of force generation and crossbridge cycle in muscle.
The purpose of this short review is to summarise some of the main findings
from such temperature-studies on mammalian muscle fibres with emphasis on our

2
T-jump experiments. It will be concluded that crossbridge force generation is en-
dothermic (is perturbed by T-jump) and is coupled to a defined kinetic step prior
to the release of inorganic phosphate (Pi) in the cycle; the temperature dependence
of steady active force is largely due to the endothermic nature of the force genera-
tion step; the strain-sensitivity of this step canprovide a basis for the well-known
Fenn-effect in muscle contraction (Fenn, 1924); structural mechanism of endo-
thermic crossbridge force generation remains unclear.
3. Materials and methods
This account refers to complementary findings from two different fast muscle
fibre experimental preparations, namely electrically activated intact fibre bundles
and chemically activated skinned fibres. Only some essential features of materials
and methods, whichwould be of interest to temperature studies, are summarized
below; comprehensive details are published elsewhere.
3.1. Mechanical Recording System
The trough system, mounted on an optical microscope stage, consisted of
three ~50 μl troughs milled in a titanium block and a front experimental trough
with glass windows in the front and bottom (Ranatunga, 1996). The temperature
of the trough system was kept <5 °C but,by Peltier modules,the front trough tem-
perature could be independently clamped at a suitable temperature. In order to re-
duce the temperature sensitivity of the force (=tension) recording, the transducer
was built using two AE 801 elements (Akers, Norway): one cut-beam element was
connected to the muscle fibre (natural resonant frequency ~14 kHz)and the other
set close-by acted as a dummy, forming a full bridge (Ranatunga, 1999a). Amov-
ing-coil motor was used to produce ramp length changes in muscle fibres.
3.2 Temperature-jump Technique
A near infra-red (λ = 1.32 μm) pulse of 200 μs duration (maximum power = 2
J per pulse) from a Nd-YAG laser (Schwartz Electro-Optics Inc., Florida, U.S.A.)
was used to induce a T-jump in the front trough. The energy absorption by water
at this wavelength (~50 %) was such that the laser pulse that entered the trough
through the front window was reflected back by the aluminium foil in the back
wall and raised the temperature of the 50 μl aqueous medium in the trough, and
the fibre immersed in it, by 3-5 degrees in 200 μs. The raised solution temperature
remained constant for ~500 ms, but the duration could be increased by using the
Peltier T-clamping (see Ranatunga, 1996).

3
3.3 Intact fibre experiments
Intact fibre experiments referred to here were performed on bundles of fibres
isolated from fast flexor hallucis brevis muscle of adult male rats (Coupland &
Ranatunga, 2003); animals were killed with an intra-peritoneal injection of an
overdose (~150 mg kg-1 body weight) of Sodium Pentobarbitone (Euthatal, Rhône
Mérieux). Small bundles of ~ 5-10 intact excitable fibres were dissected from the
mid-belly of the whole muscle and aluminium foil T-clips were fixed on the ten-
dons within 0.2 mm of the fibre-ends; resting fibre length was ~2 mm, sarcomere
length ~2.5 µm and the bundle width was 100-200 μm.
A preparation was super-fused with physiological saline solution containing
(mM) NaCl, 109; KCl, 5; MgCl2, 1; CaCl2, 4; NaHCO3, 24; NaH2 PO4, 1; sodium
pyruvate, 10 and 200 mg l-1 of bovine foetal serum. The solution was bubbled with
a mixture of 95 % O2 and 5 % CO2 and was directly stimulated with supra-
maximal voltage pulses (<0.5 ms duration) applied to two platinum plate elec-
trodes placed on either side of a bundle (see Ranatunga, 1984).
3.4 Skinned fibre experiments
Bundles of fi bres from psoas muscle of adult male rabbits (killed by an intra-
venous injection of an overdose of sodium pentobarbitone)were chemically
skinned using 0.5 % Brij 58. A segment of a single fibre (2-4 mm in length) was
mounted (using nitro-cellulose glue) between two hooks, one attached to the force
transducer and the other to the motor. The sarcomere length in a 0.5 mm region of
the fibre near the tension transducer was monitored using He-Ne laser diffraction
(see Ranatunga et al., 2002); the sarcomere length was ~2.5 m.
The buffer solutions contained 10 mM glycerol-2-phosphate as a low tem-
perature-sensitive pH buffer and also 4 % Dextran (mol. wt. ~ 500 kDa) to com-
press the filament lattice spacing in the fibres to normal dimensions; major anion
was acetate, ionic strength = 200 mM, pH =7.1. For other specific details of the
solution compositions,see Coupland et al., (2001; 2005).
3.5. Some General Considerations
When dealing with tension responses induced by different perturbations,
some difficulty is encountered in identifying the homologous components. In
keeping with the nomenclature used in our previous papers (see Vawda et al.,
1999; Coupland et al., 2001; Ranatunga et al., 2002), the following components,
or phases, may be recognised in the force transients.
Phase 1: The force change that occurs concomitant with a perturbation, where the
extreme force reached is referred to as T1, as used in the original length perturba-

4
tion experiments (Huxley & Simmons, 1971). An instantaneous drop in force
(phase 1) is also seen in T-jump (pressure-release = P-jump) experiments, proba-
bly induced by expansion in series elasticity. A T-jump or a P-jump induces a
drop in force in rigor muscle fibres and this provides support for the expansion in
some elasticity (see Goldman et al., 1987; Ranatunga et al., 1990).
Phase 2: Following phase 1, the force recovers quickly to T2 force level after a
length perturbation. This partial force recovery consists of two exponential com-
ponents (Davis & Harrington, 1993) - referred to as phase 2a (fast) and phase 2b
(intermediate) (Ranatunga et al., 2002). The force generation, i.e. force rise above
the pre-perturbation level, induced by a T-jump (and a P-jump) corresponds to
phase 2b. In T-jump and P-jump experiments wher e a prominent phase 1 was seen
(see Ranatunga, 1999b; Vawda et al., 1999), a quicker force recovery correspond-
ing to phase 2a was also seen; this phase tends to partially recover the force to pre-
perturbation level as in length perturbation. Both phase 1 and phase 2a are not
prominent in the small amplitude T-jumpsused here.
Phase 3: The slower exponential component of the force transient induced by a T-
jump (and a P-jump) in isometric state.
In summary, only phases 2b and 3 are readily seen in the isometric T-jump tran-
sients;in shortening muscle phase 3 is not seen. Hence, particular emphasis is
phase 2b (endothermic force generation).
4. Temperature dependence of isometric force
4.1 Intact muscle fibres
Fig.1A shows isometric tetanic contractions from an intact (fast) muscle fibre
bundle; the steady active force (=tension) is higher at higher temperatures. Fig. 1B
shows normalised tetanic tension data from several experiments, obtained at a
range of temperatures in warming and cooling; the data shows that the relation be-
tween force and (reciprocal) temperature is sigmoidal with a half-maximal tension
at ~10 ºC. Fig. 2 shows the tension response induced by a T-jump, applied on the
plateau of tetanus. As found in Ca-activated skinned fibres (see Davis & Harring-
ton, 1987; Goldman et al., 1987; Bershitsky & Tsaturyan, 1992; Ranatunga,
1996), a T-jump induces a biphasic tension rise that reaches a new steady level,
both at 10 and 20 ºC; for a given T-jump, the initial tension rise (phase 2b) is
faster but the amplitude is less at the higher temperature. Phase 2b is thought to
represent endothermic force generation in attached crossbridges. Studies have
shown that stiffness remains unchanged (see ref in Roots & Ranatunga, 2008) or
tension / stiffness ratio is increased (Galler & Hilber 1998) with increase of tem-
perature; additionally, single-molecule experiments of Kawai et al., (2006)
showed that the force each crossbridge generates is independent of temperature.
On the basis of such findings, it may be argued that steady active force in isomet-
ric muscle may be simplified to and treated as a two state system - pre-force gen-

5
erating (low-force) and force generating (high-force) states and,while the total
number of attached crossbridges remains the same, higher temperature favours
(endothermic) force generation.In principle, such a simplistic scheme can account
for the sigmoidal temperature dependence as being due to the sh ift in the equilib-
rium from low-to high-force states (Davis, 1998; Roots & Ranatunga, 2008).
Figure 1 A: Tetanic contractions recorded from one intact fibre bundle at four temperatures, us-
ing appropriate stimulation frequencies and durations. B: Tetanic tension data from 8 bundles are
plotted as a ratio of that at 35 ºC; abscissa is reciprocal absolute temperature. The relationship
between tetanic tension and temperature is approximately sigmoidal with a half-maximal tension
occurring at ~10 °C (adapted from Coupland & Ranatunga, 2003).
Figure 2. T-jump tension responses. A: A T-jump of ~4 ºC applied during the tetanus plateau,
at 10 ºC, induces a small instantaneous drop in tension (phase 1) followed by a rise to a new
steady level. B: The tension rise on an expanded time scale is fitted with a bi-exponential curve
and the rates for phases 2b and 3 were 37 s-1 and 9 s-1 respectively. C, D: A corresponding pair of
records from the same bundle at 20 °C. Note that the tetanic tension is higher but the tension in-
crement induced by the T-jump is smaller but faster.
0 0.5 1.0 1.5
0
50
100
150
200
250
Force(kN.m
-2
)
0
100
200
300
0 0.25 0.50 0.75
time( s)
160
180
200
220
260
280
300
10 °C
20 °C
AB
CD
100 ms
350
325
300
275
250
225
200
175
150
125
100
75
50
25
0
mN
Tension
0.6 0.8 1.0 1.2 1.4
s
100 kN.m-2
35 ºC
25 ºC
15 ºC
5 ºC
stop
stop
sto
p
200 ms
N
o
r
m
a
l
i
s
e
d
t
e
n
s
i
o
n
A
3.2 3 .3 3. 4 3.5 3. 6 3.
7
10 3K/ T
0
0.5
1.0
Normalised tension
010203040 °
C
B

6
4.2 Skinned fibres
Although recording individual contractions from a preparation in warming
and cooling and at a range of temperatures (as in intact fibres) was not feasible
with the more fragile skinned fibre preparations, use of skinned fibres enables one
to readily change the chemical composition of the intracellular medium. Thus, en-
dothermic force generation to a T-jump and sigmoidal temperature dependence of
force are not seen in rigor fibres (depleted of ATP and crossbridges attached but
not cycling), nor in relaxed fibres (crossbridges detached); due to thermal expan-
sion (Goldman et al., 1987; Ranatunga, 1996), rigor force dropped instantly with a
T-jump (phase 1) and showed no recovery and the steady rigor force decreased
linearly with increase of temperature (Ranatunga, 1994). Hence, endothermic
force arises from attached crossbridges when actively cycling.
Figure 3. A: Effect of Pi. Pooled data from five fibres in each of which tension data (with maxi-
mal Ca-activation) were collected at different temperatures (range ~5-30 ºC). A fibre was acti-
vated at ~5 ºC, and temperature raised by laser T-jumps and / or Peltier. Tensions recorded in
each contraction were normalised to control at 30 ºC, and plotted against reciprocal absolute
temperature. Filled circles and solid curve are from activation in control solution (no added Pi).
Open symbols are the means (± SD) pooled tensions in the presence of 25 mM Pi from two se-
ries (i.e. before and after control). Pi depresses tension, the relative Pi-induced tension depression
is less at the higher temperatures and the curve shifted to left (From Coupland et al., 2001). B:
Effect of ADP: Pooled data from 18 fibres where tension was measured in control solution and
with 4 mM added MgADP at one or more temperatures. Mean (± s.e.m.) specific tension (in kN
m-2) are shown for control (open symbols) and for 4 mM MgADP (filled symbols); with ADP
tension is potentiated, relative potentiation is less at higher temperatures and the curve is shifted
to right (From Coupland et al., 2005).
Under control conditions, a sigmoidal temperature dependence of maximal
active force, similar to intact fibres, is obtained in maximally Ca-activated skinned
fibres (Ranatunga, 1994); Fig. 3 from two studies (Coupland et al., 2001; 2005)
show that the position of the sigmoidal relation, with respect to temperature, is
sensitive to the levels of inorganic phosphate (Pi) and MgADP, two products re-
leased during crossbridge (acto-myosin ATPase) cycling; Pi is released earlier and
3.2 3.3 3.4 3.5 3.6 3.7
103K/T
0
0.2
0.4
0.6
0.8
1.0
Ten sion
+25-Pi
30 20 10 0°C
control
30 20 10 5 °C
Tension (kN m-2)
3.2 3.3 3.4 3.5 3.6 3.7
10
3
K/T
0
300
200
100
4 mM MgADPControl
AB
10
3
K/T
Tension
25 mM Pi
Control
10
3
K/T

7
ADP later in the cycle (see Goldman et al., 1984; Hibberd et al., 1985; He et al.,
1997; 1999). Fig. 3A shows that active force is depressed by Pi so that the curve is
shifted to higher temperatures, whereas Fig. 3B shows that MgADP potentiates
force and the curve is shifted to lower temperatures. It is also seen that, because of
the endothermic nature of force generation, the relative effects on tension at a
given level of Pi or ADP is less at higher temperature; for instance, the force de-
pression by 25 mM Pi is ~50% at ~10 ºC wher e as it is ~20% at physiological
temperatures of ~30 ºC (Fig. 3A).
5. Tension responses to temperature-jump (T-jump)
5.1 Effects of Pi and ADP
Figure 4: Effect of Pi. Tension response to a T-jump of ~3 °C after the fibre was maximally Ca-
activated to steady state at ~9 °C. A: On control activation (no added Pi) B: On reactivation in a
medium containing 12.5 mM added Pi. A bi-exponential curve is fitted to each transient. With Pi
the steady force is depressed, but the initial T-jump transient (phase 2b) is faster; the amplitude
of the tension rise is similar to control (adapted from Ranatunga, 1999a).
To determine the molecular step in the ATPase cycle that underlies endo-
thermic force generation, force transients induced by a standard T-jump and at ~9-
10 ºC were examined in control and in the presence of Pi or MgADP. Fig. 4A
shows a force transient induced by a T-jump in control and Fig. 4B shows the
force transient from the same fibre when it was subsequently reactivated in the
presence of 12.5 mM added Pi. It is seen that, compared to the control, the steady
force before and after the T-jump is lower in the presence of Pi but the initial force
rise (phase 2b or endothermic force generation) is clearly faster with Pi. Fig. 5A
shows T-jump force transient fr om a fibre in control and Fig. 5B the force tran-
sient in the presence of 4 mM MgADP. Compared to the control, the steady force
is higher but the T-jump force rise is clearly slower in the presence of ADP.
0 0 . 2 0 . 4
t i m e /s
1 5 0
1 7 5
2 0 0
2 2 5
2 5 0
Force /kN.m-2
A C o n t r o l ( n o a d d e d p h o s p h a
t
0 0 . 0 4 0 . 0 8
0
0 0 . 2 0 . 4
t i m e / s
7 5
100
125
150
B+ 1 2 . 5 m M p h o s p h a t e
0 0 . 0 4 0 . 0 8
0
B
A
Time (s) Time (s)
Force (kN/m
2
)

8
Figure 5: Effect of MgADP. A: Control (no added MgADP), protocol similar to Fig. 4. B: An
identical T-jump induced in the same fibre after reactivation in the presence of 4 mM MgADP.
Note that the pre-T-jump tension is higher but the T-jump induced tension rise is slower in the
presence of MgADP; the amplitude of the tension rise is similar (from Coupland et al., 2005).
The contrasting effects of Pi and ADP on the time course of T-jump force
transient are summarised in Fig. 6; the amplitude of tension rise remained similar
(not illustrated) and phase 3 was not much sensitive to Pi or ADP. On the other
hand, en dothermic force generation (phase 2b) becomes faster with increase of Pi
(Fig. 6A) whereas it becomes slower with increase of ADP (Fig. 6B); in both
cases the time course change saturates at higher levels.
Pi (inorganic phosphate) is released earlier in the cr ossbridge cycle and the
steady muscle force is decreased with added Pi (Cooke & Pate, 1985; Hibberd et
al., 1985) but the kinetics of the approach to the new steady state were enhanced;
this has been shown studies using different techniques, such as hydrostatic pres-
sure-release (P-jump, Fortune et el., 1991), sinusoidal length oscillation (Kawai &
Halvorson, 1991) and Pi-jump (Dantzig et al., 1992; Tesi et al., 2000) and Pi-
measurement (He et al., 1997). The unified thesis that arose from such different
studies was that Pi-release in active muscle occurs in two steps,both steps are re-
versible and, kinetically, the crossbridge force generation precedes Pi-release.
Findings from T-jump experiments are consistent with that thesis (Figs. 4, 6A) and
also show that this force generation is endothermic.
The steady active tension was potentiated when [Mg.ADP] is increased; the
binding of MgADP to nucleotide-free crossbridges (AM) leading to accumulation
of force-bearing AM-ADP states (Cooke & Pate, 1985; Dantzig et al., 1991; Lu et
al., 1993; 2001; Seow & Ford, 1997), in general, may underlie the tension in-
crease. When [MgADP] is increased, the tension rise induced by a T-jump was
slower (Figs. 5, 6B) indicating that the approach to the new steady state at the
post-T-jump temperature is slower.
0 0.1 0.2 0.3 0.4 0.5
Time (s)
110
120
130
140
150
Tension (kN/m
2
)
0 0.1 0.2 0.3 0.4 0.5
Time (s)
140
150
160
170
180
AB

9
Figure 6. From experiments as in Fig. 4, 5 (T-jump from ~9 -12 ºC), the mean (± s.e.m.) rate
constants for phase 2b (filled symbols) and phase 3 (open symbols) are shown. A: Pi-
dependence: Phase 2b rate increases with Pi, exhibits saturation at high Pi levels and the relation
is hyperbolic (the curve fitted to the data). Phase 3 shows minimal sensitivity to Pi and may rep-
resent contribution to force rise of crossbridges going through a slower step in the cycle after Pi-
release (adapted from Ranatunga, 1999). B: ADP dependence: Phase 2b rate decreases with
ADP, exhibits saturation at high ADP levels and the relation is hyperbolic. Phase 3 shows mini-
mal sensitivity to ADP (adapted from Coupland et al., 2005)
5.2 Effect of strain (during shortening and lengthening)
Temperature perturbation experiments referred to above were on isometric
muscle fibres, whereas it is known that the force that a muscle develops varies
with velocity of filament sliding in their sarcomeres, i.e. during steady muscle
shortening and lengthening. With increase of shortening velocity, force declines
below isometric force (P0) and reaches zero at the maximum velocity; conversely,
an active muscle develops ~2 x P0as lengthening velocity is increased to 1-2 L0
(muscle fibre length) /s (Hill, 1938; Katz, 1939; Lombardi & Piazzesi, 1990).
Moreover, the en ergy production and the acto-myosin ATPase rate in muscle are
increased with shortening and decreased with lengthening, (Fenn, 1924; Curtin &
Davies, 1973; Getz et al., 1998; He et al., 1999; Linari et al., 2003). Hence,in a
recent study, in addition to recording under isometric condition as before, we also
examined the tension response induced by a T-jump in maximally active muscle
fibres, wh en their force was decreased to a steady level by ramp shortening or in-
creased to a steady level by ramp lengthening. The range of velocities used was 0-
0.2 L0 /s and the unloaded (maximum) shortening velocity at this temperature was
~1L0/s; force decreased to <0.5 x P0 when shortening at 0.2 L0/s and increased to
2-3 x P0 when lengthening at >0.05 L0/s (for details, see Ranatunga et al., 2007)
01234
0
10
20
30
40
50
MgADP (mM)
0 10 20 30
[Pi] mM
25
75
125
Phase 2b (s
-1
)
5
10
Phase 3 (s
-1
)
AB

10
AB
C
A
4
0
-4
%L
0
A
B
Figure 7: A: A fibre held isometric was maximally Ca-activated at ~9 ºC and, during the tension
plateau (P0), a T-jump of ~3 ºC applied (top panel - schematic) to obtain the “isometric” tension
trace; bottom panel shows length records. Temperature was clamped again at ~9 ºC and fibre
stretched at a constant velocity to obtain the “lengthening” tension traces (one without and the
other with a T-jump); during lengthening, the tension rises to ~2.2 P0 and T-jump does not lead
to a net increase of tension, but induces an obvious the instantaneous tension drop (phase 1). The
procedure was then repeated with fibre shortening to obtain “shortening” tension traces; tension
decreases to ~0.5 P0, but T-jump induces a pronounced tension rise; the post T-jump tension
trace (300ms)is fitted with a single exponential function. B and C:Data from 6 fibres, velocity
is plotted as L0/s on the abscissa, negative for shortening and positive for lengthening. B: The net
tension change after a T-jump is plotted as a ratio of the post-T-jump tension. During shortening
(filled circles) T-jump tension rise is correlated with velocity (P<0.05); each symbol is a mean
(±s.e.m., n=7,8) and curve fitted by eye. During lengthening, the tension change (open symbols,
individual data) is not significantly different from zero. Mean (s.e.m., n=30) for isometric state is
plotted on the ordinate. C: With shortening, rate of tension rise (mean ±s.e.m.) is correlated ve-
locity (P<0.001). During len gthening, rate of tension rise from curve fit to the late part of pre-T-
jump tension trace (crosses)is not significantly different (P>0.1) from the post-T-jump rate
(open circles). Two isometric rates are on the ordinate (adapted from Ranatunga et al., 2007).
Superimposed tension responses from a fibre in Fig. 7A illustrate the T-jump
tension responses (middle panel) in the different mechanical states. During ramp
lengthening (top tension traces), the tension rises towards a level of about twice
the isometric tension (P0) and a T-jump produces no further tension rise; a small

11
instantaneous drop in tension was some times seen, indicative of thermal expan-
sion in the fibre (see Ranatunga, 1996). During steady shortening (lower traces),
the tension decreases to a level lower than P0, and a T-jump produces a marked
(mono-phasic) tension rise. As given above, a T-jump induces a biphasic rise in
tension when the fibre is held in isometric (middle trace). Fig. 7B shows that, plot-
ted as a ratio of the post-T-jump tension, the amplitude of the T-jump tension rise
in shortening fibres is higher than isometric and is correlated with velocity (filled
circles); the amplitude of tension ch ange during lengthening is not different from
zero. Fig. 7C show that the rate of T-jump-induced tension rise during steady
shortening increases linearly with velocity. For completeness, the data for the
post-T-jump tension rise during steady lengthening are also shown (open circles).
Since the amplitude is negligible (Fig. 7B), this is only an apparent rate and it in-
creases slightly with velocity; the rates of tension rise determined by curve fit to
the late part of pre-T-jump tension trace (crosses) were not significantly different
from the post-T-jump rates (Students t-test, P>0.05). As given above, the T-jump-
induced tension rise in the isometric case contained two components; their rates
(40-50 s-1 and 5-10 s-1) are shown by open squares on the ordinates. Thus, the data
in Fig. 7 shows that the tension during steady lengthening is not changed by a T-
jump, whereas the tension in shortening is particularly enhanced by a T-jump.
5.3. Kinetic simulation of basic findings
Incorporation of the above features to an adapted Lymn & Taylor (1971) cy-
cle leads to a minimal, 5-step, kinetic scheme for the crossbridge / AM-ATPase
cycle (Scheme 1) that can qualitatively simulate some of the findings above (see
Coupland et al., 2005; Ranatunga et al., 2007).
Scheme 1
Pi ADP (ATP)
States I II(F) III(F) IV(F) V
AM.ADP.Pi AM*.ADP.Pi  AM*.ADP AM*'.ADP  [AM]
Steps (1) (2) (3) (4)
(5)
Briefly, the forward rate constant (k+1) of Step 1 is endothermic force genera-
tion, step 2 is Pirelease / binding (similar to Dantzig et al, 1992); steps 3 and 4
represent the slow, two-step, ADP release: k+3 / k-3 was forward biased (Dantzig et
al, 1991). Step 5 is irreversible and includes all the necessary steps after ADP re-
lease to reprime a crossbridge for the next cycle. The overall rate in this route is
low (k+5, ~10 s-1), probably limited by the M.ATP↔M.ADP.Pi cleavage step after
detachment (see He et al., 2000). The linear kinetic scheme 1 above was solved by
the matrix method using Mathcad 2000 software (Mathsoft) as described previ-
ously (Gutfreund & Ranatunga, 1999). According to the scheme, states II, III and

12
IV (AM*.ADP.Pi, AM*ADP and AM*’.ADP) are equal-force bearing states (F)
and the sum of their fractional occupancy is taken as force.
Figure 8. Model simulations - isometric. A: Arrhenius plots of the two rate constants of the
model (see Scheme 1) that were changed to simulate temperature effects; the rate of the force
generation step (k+1) increased markedly (see Zhao & Kawai, 1994; Q10 ~4) and that of ADP-
release step (k+4) slightly (Q10 of 1.3). B:Filled circles show simulated, steady state tension
(sum of the fractional occupancies of states II, III and IV) under control conditions (0.5 mM Pi
and 10 μM ADP) at 5 ºC intervals. Note the approximate sigmoidal relation with half-maximal
at ~10-12 ºC. Open symbols with dotted curves show that with simulated 25 mM Pi and 4 mM
[MgADP], the sigmoidal relation is shifted to the left and right, respectively. C: Simulated ten-
sion transients to T-jump at ~10 ºC in control (solid curve) and in the presence of 30 mM Pi (dot-
ted curve); tension rise is faster with Pi. D: Tension transients, in control (solid curve) and in the
presence of MgADP (dotted curve); the tension rise is slower but amplitudes are similar with
MgADP. (In both C and D, the tension records with Pi or ADP were shifted vertically for super-
imposition) – adapted from Coupland et al., (2005).
As shown in Fig. 8A, temperature effect was simulated by increase of the rate
constant k+1 (force generation, Q10 of 4) and a small increase in step k+4 (Q10 of
1.32), since there is evidence that ADP release itself is temperature-sensitive (Se-
mankowski et al., 1985). The sum of the occupancies of states II, III and IV at ~1
s was taken as steady tension. Fig. 8B filled circles show the control steady state
tension and its distribution approximates a sigmoidal relation with half-maximal at
~10-12 ºC. Within a temperature range of ~0 to 40 ºC, the relation is shifted up-
wards along the tension axis (potentiation) and slightly to the right with 4 mM
added ADP and shifted down (depression) and to the left (to higher temperatures)
with Pi. Fig.8C and 8D show simulated tension responses induced by a standard
T-jump at ~10 ºC under control conditions (continuous trace) and with Pi (Fig.

13
8C) or MgADP (Fig. 8D); in each case, the two traces are vertically shifted for su-
perimposition to show that they have similar amplitudes; the tension rise is faster
with Pi but slower with ADP. Fig. 9A shows that, during simulated sh ortening, a
T-jump tension response is faster at higher velocity and Fig. 9B shows that, at a
given velocity, T-jump tension response becomes faster at higher temperatures.
Figure 9: Simulation –shortening: Simulated tension responses to a T-jump during steady
shortening using scheme 1, where force is normalised to that in isometric case (i.e. P0 at 10 ˚C)).
In each case, a shortening was simulated by increasing k+4 and, at steady state, a 5 ˚C T-jump (re-
setting k+1) is induced at time zero (see Gutfreund & Ranatunga, 1999). A: Simulated tension re-
sponses to T-jumps, at ~10 ˚C; initial force is reduced by shortening at different velocities (low
to high) and a T-jump induces a faster tension rise at higher velocities. B: T-jump tension re-
sponses, when shortening at the same velocity (constant k+4) at different temperatures (5 - 25 ºC).
The pre- and post- T-jump tension and the rate of tension rise to a T-jump are increased at higher
temperatures, as in experiments (adapted from Ranatunga et al., (2007).
The model is simplistic,used a minimal kinetic scheme for actin-myosin-
ATPase pathway and did not have strain-sensitive features; hence, it could only
qualitatively account for endothermic force in isometric and shortening muscle.
6. General Discussion
Findings from various temperature-studies on intact and skinned muscle fi-
bres, briefly referred to in this review, provide a useful overall picture for the
process and mechanism of muscle contraction. Thus, a fundamental characteristic
of active muscle is that its active force is endothermic (force rises on absorption of
heat); this is largely due to the force generation by an attached crossbridge itself
being temperature-sensitive. The particular molecular step is identified as being
before phosphate release in a linear acto-myosin ATPase pathway, it is enhanced
during muscle shorten ing and depressed during muscle lengthening and hence s-
train-sensitive;as summarised below, this overall thesis that has emerged from
temperature studies is broadly consistent with most findings reported from other
kinetic and mechanics studies on muscle.
1.0
0.75
0.5
0.25
Force
A
~10 ˚C, dif velocities
0 0.1 0.2 0.3
Time /s
low
high
0.75
0. 5
0.25
0.0
B
one velocity, dif temperatures
0 0.1 0.2 0.3
Time /s
25
15
5 ˚C
A B
force
Time (s)
25 ºC
5 ºC
low
high

14
6.1 Comparison with other studies
As mentioned with results, the notion that force generation step is prior to Pi-
release (a transition between two A-M.ADP.Pi states)is well supported from sev-
eral studies and using different techniques, such as hydrostatic pressure-release (P-
jump, Fortune et el., 1991), sinusoidal length oscillation (Kawai & Halvorson,
1991) and Pi-jump (Dantzig et al., 1992; Tesi et al., 2000) and Pi-measurement
(He et al., 1997). In a detailed examination of mechano-kinetic models, Smith &
Sleep (2004) indeed ruled out the alternative possibility that force generation fol-
lows Pi-release. Findings from T-jump experiments are consistent with that thesis
(Figs. 4, 6A) and also show that this force generation is endothermic (and entropy
driven),as has indeed been emphasized in other studies (Davis, 1998; Davis &
Epstein, 2007; Kawai, 2003; Zhao & Kawai, 1994).
Following the original experiments and formulations by Huxley & Simmons
(1971), quick tension recovery (T1-T2 transition) induced by a small length-release
step is thought to represent crossbridge power stroke or force generation in mus-
cle; quick tension recovery can be resolved in to two components (Davis & Har-
rington, 1993), labeled here as phase 2a and 2b. The difficulties in correlating T-
jump force generation with quick tension recovery after length-release have been
raised in previous studies (see Bershitsky et al., 1997). It is found that the T-jump
force generation is much slower than tension recover y from length release; how-
ever, in the same preparation and at similar temperature, it is faster than phase 2b
recovery from stretches (see Ranatunga et al., 2002), indicating that T-jump force
rise monitors force generation in crossbridges unstrained by filament sliding (as in
length steps); its rate (phase 2b recovery) would be enhanced in length release and
decreased following stretch, due to particular strain-dependence of the underlying
rate constants (Huxley & Simmons, 1971; Ford et al., 1977). Indeed, by extrapola-
tion, the phase 2b recovery rate that corresponds to the isometric point in a length
step versus rate of tension recovery plot in experiments on rabbit psoas fibres at
~10 ºC is ~40-60 s-1 (Ranatunga et al., 2002; Coupland et al., 2005) which is com-
parable to the rate of endothermic force generation in the T-jump experiments; the
data also indicated that this extrapolated “isometric phase 2b recovery” from
length step experiments is enhanced with Pi and depressed with MgADP, as does
T-jump force generation. In general, these observations support the thesis that
phase 2 tension recovery after a length step contains a component (phase 2b) that
is homologous to the endothermic force generation observed after a rapid T-jump,
as originally proposed by Davis & Harrington (1993; see also Davis, 1998). This
notion gains support from the study of Gilbert & Ford (1988) that showed experi-
mentally that quick tension recovery from length-release is associated with heat
absorption. Additionally, the rate of phase 2 tension recovery from length-release
is temperature sensitive (Q10 of 2-3, Piazzesi et al, 2003), the temperature sensitiv-
ity being greater for phase 2b than for phase 2a component of recovery (Davis &
Harrington, 1993; Davis & Epstein, 2003). Thus, like the T-jump force rise, phase
2b tension recovery from length-release represents an en dothermic pr ocess; ques-
tion remains as to the kinetic basis of (fast) phase 2a.

15
Despite the above considerations, however, some fibre mechanics experi-
ments of Bershitsky & Tsaturyan (2002) indicate that different pr ocesses underlie
tension responses to T-jump and length steps; Ferenczi et al., (2005) considered
apparent strain independent, but temperature-dependent, rate-limiting step prior to
force generation. Also, whereas T-jump studies above required only one molecular
step for force generation, it has been argued from on X-ray diffraction studies us-
ing length perturbation that, several molecular steps would be required to com-
plete a working stroke of a crossbridge (Huxley et al., 2006).
6.2 Mechanism of endothermic force generation
A conformational change of the acto-myosin crossbridge, resulting in lever
arm tilting, is thought to be the molecular-structural mechanism of force muscle
generation (see review Geeves & Holmes, 1999); whether this is endothermic and
how it is associated with increased entropy (disorder) remain unclear. Zhao &
Kawai (1994) proposed that altered hydrophobic interaction between actin and
myosin-head for the large increase of entropy (see also, Kodama, 1985); the tran-
sition of non-stereo-specifically attached to stereo-specifically attached cr oss-
bridge states would represent such an event, since stereospecific interaction may
be hydrophobic. Ferenczi et al., (2005) proposed such a mechanism as a force
generating step that is additional to the lever arm tilting step in the crossbridge cy-
cle (see). Davis & Epstein (2007) proposed an alternative temperature-sensitive
mechanism, namely, a local unfolding within the crossbridge secondary/tertiary
structure and suggested that this might cause the lever arm tilting movement and
force generation. From analyses of the rate of tension rise induced by T-jumps at
different temperatures as Arrhenius plots, they showed that the observed rate
could be resolved into the forward and reverse rate constants for force generation,
where the reverse rate constant showed anti-Arrhenius behaviour (negative tem-
perature-sensitivity) characteristic for protein folding. Specific experiments aimed
at getting direct information are required to elucidate the molecular mechanism.
Studies on myosin-ATPase in solution (Kodama, 1985; Millar et al., 1987)
have shown that the ATP cleavage step (i.e. in detached crossbridges in fibres) is
endothermic and evidence suggests that this is due to a temperature-dependent
conformational change(s) in myosin head (Werner et al., 1999; Malnasi-
Csizmadia et al., 2000); such findings from myosin and myosin-ATPase cycle
need to be accommodated in the acto-myosin cycle in muscle fibres.
6.3 Some implications
In principle, the active isometric muscle force is maintained by an “equilib-
rium” between low- and high-force attached crossbridge states so that a small

16
length-release (a shor tening) inducing negative strain and reducing force in at-
tached crossbridges would perturb the equilibrium leading to force generation
(Huxley & Simmons, 1971; Ford et al., 1977). Conversely, a rapid stretch (a
lengthening), inducing positive strain on crossbridges would lead to an inhibition /
reversal of the force generation. Implication is that the force generating acto-
myosin conformational ch ange is more likely to occur when exposed to negative
strain than wh en exposed to positive strain. Since T-jump perturbs an early mo-
lecular step, the T-jump tension response in active muscle can be used as a “signa-
ture” of the acto-myosin ATPase cycle. Hence, the enhanced endothermic force
generation suggests that the ATPase cycle proceeds more readily during muscle
shortening, consistent with studies on muscle energetics (see Smith et al., 2005).
The steady state force-shortening velocity curve of mammalian muscle was very
temperature sensitive such that the maximal mechanical power output increased
markedly in warming from 10 to 35 ˚C (Ranatunga, 1984; 1998); the finding that
the tension in shortening muscle is particularly sensitive to a T-jump suggests that
endothermic nature of force generation is a major underlying cause.
T-jump force generation is not seen during lengthening, indicating that the
myosin motor fails to undergo the force-generating transition and, hence, the
crossbridge / acto-myosin ATPase cycle is short-circuited before phosphate-
release and before energy liberation. An implication is that the high force in
lengthening muscle arises from the pr e-stroke crossbridges attaching, getting
strained by stretch and detaching without proceeding through the ATPase cycle, a
conclusion consistent with previous studies on muscle fibres (Lombardi & Pi-
azessi, 1990; Getz et al., 1998; Pinniger et al., 2006) and myofibrils (Rassier,
2008). The fact that the lengthening muscle tension is insensitive to a T-jump is
consistent with similar eccentric forces measured at high and low temperatures in
human muscle experiments (De Ruiter & De Haan, 2001). The contrasting behav-
iour of shortening and lengthening muscle to a T-jump suggests a basis for the
Fenn effect (Fenn, 1924),a cardinal principle of muscle contraction,that energy
liberation in muscle is enhanced during shortening and depressed during lengthen-
ing. Also, the non-endothermic nature of steady tension during lengthening and a
more obvious thermal-expansion effect by a T-jump indicate that stretch of non-
crossbridge elements within muscle may contribute to the force and energy stor-
age in lengthening muscle (Edman & Tsuchiya, 1996; Pinniger et al., 2006).
Accumulation of products of ATP hydrolysis, particularly Pi, is commonly
considered as contributory to in situ muscle fatigue. Temperature studies (e.g. Fig.
3) show that, because of the endothermic nature of force generation and that it is
not directly coupled to release of Pi or ADP (products of hydrolysis), the relative
effects on force of product accumulation would be less at high physiological tem-
peratures; observations in support of this suggestion have been reported from fa-
tigue-experiments on intact rat fast muscle fibres (see refs in Roots et al., 2009).

17
7. Acknowledgements
We thank the Wellcome Trust Foundation for financial support of our re-
search, Dr. Gerald Offer (Bristol) for valuable discussions and Blackwell Publish-
ing and Springer for permission to include data we had published in the Journal of
Physiology and the Journal of Muscle Research and Cell Motility.
8. References
Bershitsky SY & Tsaturyan AK (1992). Tension responses to joule temperature jump in skinned
rabbit muscle fibres. J Physiol 447, 425–448.
Bershitsky SY & Tsaturyan AK (2002). The elementary force generation process probed by tem-
perature and length perturbations in muscle fibres from the rabbit. J Physiol 540, 971–988.
Bershitsky SY, Tsaturyan AK, Bershitskaya ON, Mashanov GI, Brown P, Burns R & Ferenczi
MA (1997). Muscle force is generated by myosin heads stereospecifically attached to actin.
Nature 388, 188–190.
Cooke, R. & Pate, E. (1985). The effects of ADP and phosphate on the contraction of muscle fi-
bers. Biophysical Journal 48, 789-798.
Coupland ME, Pinniger, GJ & Ranatunga KW (2005). Endothermic force generation, tempera-
ture-jump experiments and effects of increased [MgADP] in rabbit psoas muscle fibres. J
Physiol 567, 471-492.
Coupland ME, Puchert E & Ranatunga KW (2001). Temperature dependence of active tension in
mammalian (rabbit psoas) muscle fibres: effect of inorganic phosphate. J Physiol 536, 879–
891.
Coupland ME & Ranatunga KW (2003). Force generation induced by rapid temperature jumps in
intact mammalian (rat) muscle fibres. J Physiol 548, 439-449.
Curtin NA & Davies RE (1973). Chemical and mechanical changes during stretching of acti-
vated frog skeletal muscle. Cold Spring Harb Symp Quant Biol 37, 619-626.
Dantzig JA, Goldman YE, Millar NC, Lacktis J & Homsher E (1992). Reversal of the cross-
bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fi-
bres. J Physiol 451, 247-278.
Dantzig JA, Hibberd MG, Trentham DR & Goldman YE (1991). Crossbridge kinetics in the
presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J
Physiol 432, 639-680.
Davis JS (1998). Force generation simplified. Insights from laser temperature-jump experiments
on contracting muscle fibres. In Mechanisms of Work Production and Work Absorption in
Muscle, ed. Sugi H & Pollack GH, pp. 343–352. Plenum Press, New York.
Davis JS & Epstein ND (2007). Mechanism of tension generation in muscle: an analysis of the
forward and reverse rate constants. Biophys J 92, 2865-2874.
Davis JS & Harrington W (1987). Force generation by muscle fibers in rigor: a laser tempera-
ture-jump study. Proc Natl Acad Sci U S A 84, 975–979.
Davis JS & Harrington W (1993). A single order-disorder transition generates tension during the
Huxley-Simmons phase 2 in muscle. Biophys J 65, 1886–1898.
De Ruiter CJ, Jones DA, Sargeant AJ & De Haan A (1999). Temperature effect on the rates of
isometric force development and relaxation in fresh and fatigued human adductor pollicis
muscle. Exp Physiol 84, 1137–1150.
Edman KA & Tsuchiya T (1996). Strain of passive elements during force enhancement by
stretch in frog muscle fibres. J Physiol 490, 191–205.
Fenn WO (1924). The relationship between the work performed and the energy liberated in mus-
cular contraction. J Physiol 59, 373-395

18
Ferenczi MA, Bershitsky SY, Koubassova N, Siththanandan V, Helsby WI, Pannie P, Roessle
M, Narayanan T & Tsaturyan AK (2005). The ‘Roll and Lock’ mechanism of force genera-
tion in muscle. Structure 13, 131–141.
Ford LE, Huxley AF & Simmons RM (1977). Tension responses to sudden length change in
stimulated frog muscle fibres near slack length. J Physiol 269, 441–515. 448
Fortune, N.S., Geeves, M.A. & Ranatunga, K.W. (1991). Tension responses to rapid pressure re-
lease in glycerinated rabbit muscle fibers. Proceedings of National Academy of Science 88,
7323-7327.
Galler S & Hilber K (1998) Tension/stiffness ratio of skinned rat skeletal muscle fibre types at
various temperatures. Acta Physiol Scand 162, 119-126.
Geeves MA & Holmes KC (1999). Structural mechanism of muscle contraction. Ann Rev Bio-
chem 68, 687-728.
Getz, EB, Cooke R & Lehman SL (1998) Phase transition in force during ramp stretches of
skeletal muscle. Biophys J 75, 2971-2983.
Gilbert SH & Ford LE (1988). Heat changes during transient tension responses to small releases
in active frog muscle. Biophys J 54, 611-677.
Goldman YE, Hibberd MG & Trentham DR (1984). Relaxation of rabbit psoas muscle fibres
from rigor by photochemical generation of adenosine-5’-triphosphate. J Physiol 354, 577-
604.
Goldman YE, McCray JA & Ranatunga KW (1987). Transient tension changes initiated by laser
temperature jumps in rabbit psoas muscle fibres. J Physiol 392, 71-95.
Gutfreund H & Ranatunga KW (1999). Simulation of molecular steps in muscle force genera-
tion. Proc Roy Soc B 266, 1471-1475.
Hadju S. (1951). Behaviour of frog and rat muscle at higher temperatures. Enzymologia 14, 187-
190.
He, Z-H, Chillingworth RK, Brune M, Corrie JET, Trentham DR, Webb MR & Ferenczi MA
(1997). ATPase kinetics on activation of rabbit and frog permeabilised isometric muscle fi-
bres: a real time phosphate assay. J Physiol 501, 125-148.
He, Z-H, Chillingworth RK, Brune M, Corrie JET, Webb MR & Ferenczi MA (1999). The effi-
ciency of contraction in rabbit skeletal muscle fibres, determined from the rate of release of
inorganic phosphate. J Physiol 517, 839-854.
He, Z-H, Bottinelli R, Pellegrino MA, Ferenczi MA & Reggiani C (2000). ATP consumption
and efficiency of human single muscle fibers with different myosin isoform composition.
Biophys J 79, 945-961.
Hibberd MG, Dantzig JA, Trentham DR & Goldman YE (1985). Phosphate release and force
generation in skeletal muscles fibers. Science 228, 1317-1319.
Hill AV (1938). The heat of shortening and the dynamic constants of muscle. P Roy Soc Lond B
126, 136-195.
Huxley AF (1957). Muscle structure and theories of contraction. Prog Biophys 7, 285–318.
Huxley AF & Simmons RM (1971). Proposed mechanism of force generation in striated muscle.
Nature 233, 533–538.
Huxley HE (1969). Mechanism of muscle contraction. Science 164, 1356-1366.
Huxley HE, Reconditi M, Stewart A & Irving T (2006). X-ray interference studies of crossbridge
action in muscle contraction: evidence from muscles suring steady shortening. J Mol Biol
363, 762-772.
Katz B (1939). The relations between force and speed in muscular contraction. J Physiol 96, 45–
64.
Kawai M (2003). What do we learn by studying the temperature effect on isometric tension and
tension transients in mammalian striated muscle fibres? J Musc Res Cell Motil 24, 127-138.
Kawai M & Halvorson HR (1991). Two step mechanism of phosphate release and the mecha-
nism of force generation in chemically skinned fibers of rabbit psoas muscle. Biophys J 59,
329–342.

19
Kawai M, Kido T, Vogel M, Fink RHA, & Ishiwata S. (2006). Temperature change does not af-
fect force between regulated actin filaments and heavy meromyosin in single molecule ex-
periments. J Physiol 574, 877-878.
Kodama T (1985). Thermodynamic analysis of muscle ATPase mechanisms. Physiol Rev 65,
467-551.
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 stimu-
lated frog muscle fibres. J Physiol 431, 141-171.
Lu Z, Moss RL & Walker JW (1993). Tension transients initiated by photogeneration of MgADP
in skinned skeletal muscle fibers. J Gen Physiol 101, 867-888.
Lu Z, Swartz DR, Metzger JM, Moss RL & Walker JW (2001). Regulation of force development
studied by photolysis of caged ADP in rabbit skinned psoas fibers. Biophys J 81, 334-344.
Lymn RW & Taylor EW (1971). Mechanism of adenosine triphosphate hydrolysis by actomy-
osin. Biochem 10, 4617-4624.
Malnasi-Csizmadia A., Woolley RJ & Bagshaw CR (2000). Resolution of conformational states
of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: Implications for
the open-closed transition identified by crystallography. Biochem 39, 16135-16146.
Millar NC, Howarth JV & Gutfreund H (1987). A transient kinetic study of enthalpy changes
during the reaction of myosin subfragment 1 with ATP. Biochem J, 248, 683-690.
Piazzesi G, Reconditi M, Koubassova N, Decostre V, Linari M, Lucil L & Lombardi V. (2003).
Temperature dependence of the force-generating process in single from the frog skeletal
muscle. J Physiol 549, 93-106.
Pinniger G J, Ranatunga KW and Offer GW (2006). Crossbridge and non-crossbridge contribu-
tions to tension in lengthening rat muscle: force-induced reversal of the power stroke. J
Physiol 573, 627–643.
Ranatunga KW (1984). The force–velocity relation of rat fast- and slow-twitch muscles exam-
ined at different temperatures. J Physiol 351, 517–529.
Ranatunga KW (1994). Thermal stress and Ca-independent contractile activation in mammalian
skeletal muscle fibers at high temperatures. Biophys J 66, 1531–1541.
Ranatunga KW (1996). Endothermic force generation in fast and slow mammalian (rabbit) mus-
cle fibers. Biophys J 71, 1905–1913.
Ranatunga KW (1998). Temperature dependence of mechanical power output in mammalian
(rat) skeletal muscle. Exp Physiol 83, 371-376.
Ranatunga, K.W. (1999a). Effects of inorganic phosphate on endothermic force generation in
muscle. Proc R Soc B266, 1381-1385.
Ranatunga, K.W. (1999b). Endothermic force generation in skinned cardiac muscle from rat. J
Musc Res Cell Motil 20, 489-490.
Ranatunga KW, Coupland ME & Mutungi G (2002). An asymmetry in the phosphate depend-
ence of tension transients induced by length perturbation in mammalian (rabbit psoas) muscle
fibres. J Physiol 542, 899–910.
Ranatunga KW, Coupland ME, Pinniger GJ, Roots, H & Offer GW (2007). Force generation ex-
amined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas)
muscle fibres. J Physiol 585, 263-277
Ranatunga, K.W., Fortune, N.S. & Geeves, M.A. (1990). Hydrostatic compression in glyceri-
nated rabbit muscle fibres. Biophys. J 58, 1401-1410.
Ranatunga KW & Wylie SR (1983). Temperature-dependent transitions in isometric contractions
of rat muscle. J Physiol 339,87–95.
Rassier DE. (2008). Pre-power stroke cross bridges contribute to force during stretch of skeletal
muscle myofibrils. Proc R Soc B275, 2577-2586.
Roots H, Ball G, Talbot-Ponsonby J, King M, McBeath K & Ranatunga KW (2009). Muscle fa-
tigue examined at different temperatures in experiments on intact mammalian (rat) muscle fi-
bers. J Appl Physiol (In Press).

20
Roots H & Ranatunga KW (2008). An analysis of temperature-dependence of force, during
shortening at different velocities, in (mammalian) fast muscle fibres. J Musc Res Cell Motil
29, 9-24.
Seow CY & Ford LE (1997). Exchange of ADP on high-force cross-bridges of skinned muscle
fibers. Biophys J 72, 2719-2735.
Siemankowski RF, Wiseman MO & White HD (1985). ADP dissociation from actomyosin sub
fragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle.
Proc Nat Acad Sci USA 82, 658-662.
Smith NP, Barclay CJ, Loiselle DS (2005). The efficiency of muscle contraction.Prog Biophys
& Mol Biol 88, 1-58.
Smith GA & Sleep J (2004). Mechanokinetics of rapid tension recovery in muscle: the myosin
working stroke is followed by a slower release of phosphate. Biophys J 87 442-456.
Tesi C, Colomo F, Nencini S, Piroddi N & Poggesi C (2000). The effect of inorganic phosphate
on force generation in single myofibrils from rabbit skeletal muscle. Biophys J 78, 3081-
3092.
Vawda F, Geeves MA & Ranatunga KW (1999). Force generation upon hydrostatic pressure re-
lease in tetanized intact frog muscle fibres. J Musc Res Cell Motil 20, 477–488.
Werner J, Urbanke C & Wray J (1999). Fluorescence temperature-jump studies of myosin S1
structures. Biophys J 76, M-AM-G8.
Zhao Y & Kawai M (1994). Kinetic and thermodynamic studies of the cross-bridge cycle in rab-
bit psoas muscle fibers. Biophys J 67, 1655-1668.