The efficiency of contraction in rabbit skeletal muscle fibres ...

J. Physiol. 517.3 Efficiency of muscle contraction
841
obtained from Coherent-Ealing Ltd, Watford, UK) and focused by
a60mm FL plano-convex lens onto the plane of the photodiode
described below. The beam was 1 mm high, extended 2·1 mm along
the fibre and illuminated the fibre normally, in a plane at 5 deg to
that of the frequency-doubled light so that the non-diffracted beam
emerged from the fibre through the front window below the
frequency-doubled beam. For a sarcomere length of 2·7 ìm, the
light from each of the first orders of diffraction emerged at an angle
of 13·55 deg with respect to the zero order. As the scattering effect
of the fibre spread the diffracted beam in the vertical plane, a biconvex
cylindrical lens (40 mm FL made up of two plano-convex
80 mm FL lenses) was used to collect each diffracted beam and to
focusittoaspotontooneoftwo9mm-long position-sensitive
photodiodes (PS-100-10; Quantrad, Santa Clara, CA, USA). An
electrical signal was obtained from each edge of the positionsensitive
diodes. The sum of these signals indicated the intensity of
the light falling on the photodiode whereas the difference was
sensitive to the position of the diffraction spot. The ratio of the
difference to the sum of the output signals was used in the
measurements as it varies linearly with the position of the centroid
of the light falling on the diode. The degradation of the diffraction
signal was shown by a decrease in the summed signal. The lenses
and photodiodes were mounted on an horizontal arc centred on the
middle of the fibre at a radius of 170 mm. Micrometer screws
allowed each photodiode to be moved horizontally, tangentially to
the circular track, with 10 ìm precision. For each fibre, only the
brighter of the two diffracted first-order beams was used. At the
beginning of each experiment, the photodiode signal was adjusted
to zero by moving the photodiode along the track to centre the
diffraction spot and the corresponding sarcomere length of the fibre
was measured through the microscope using bright-field
illumination with a ² 40 0·75 NA water-immersion objective lens
and a ² 10 eyepiece (both from Zeiss). After adjustment of the
electrical signal to zero, calibration of the sarcomere length signal
was achieved for each fibre by recording the photodiode electrical
signal in response to movement of the photodiode by a known
distance along the track. The change in electrical signal achieved by
moving the photodiode, for example, by 1 mm in the direction
away from the zero order was equivalent to the change in electrical
signal resulting from movement of the diffraction spot by the
sarcomeres shortening from a length of 2·70 ìm to 2·64 ìm. The
sarcomere signal was sharpest and brightest in fresh fibres, and
deteriorated after each photolytically induced contraction. Some
recovery of the signal occurred during the relaxation phase after
each contraction. The sarcomere signal also gradually deteriorated
during each contraction, so that in some cases, only the first few
hundred milliseconds following photolytic release of ATP produced
reliable measurements of sarcomere length. In some traces, a slow
drift in the sarcomere signal can be attributed to deterioration of
the sarcomere signal rather than to sarcomere shortening, as
evidenced by an accompanying decrease in the intensity of the
sarcomere signal. The slow drift seen in Fig. 1D after the period of
steady shortening is an example of this.
Data collection
Fluorescence, force, motor output signal and sarcomere length
signals were collected using a 12-bit analog-to-digital circuit
operated at a minimum of 1 kHz (Computerscope, R.C. Electronics
EGAA Computerscope, Goletta, CA, USA in an Intel Pentium
133 MHz computer). A chart recorder continuously monitored the
force and fluorescence signal as a means of evaluating the state of
the fibres. The fluorescence signal was converted into the amount of
Pé released by the fibres as explained below.
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Solutions
The experimental solutions were described previously (He et al.
1997) and consisted of ‘relaxing’, ‘activating’, ‘loading’ and ‘rigor’
solutions. The ionic strength of all solutions was calculated to be
0·15 Ò. The pH was adjusted to 7·1 at 20 °C. Care was taken to
minimize contamination of the solutions with Pé.
Relaxing solution consisted of 60 mÒ TES, 10 mÒ EGTA, 1 mÒ
free Mg¥, 5 mÒ MgATP (6·2 mÒ total ATP), 10 mÒ glutathione,
with ionic strength adjusted with potassium propionate. Calciumfree
rigor solution was identical to relaxing solution, except for the
omission of ATP and contained 4 units ml¢ apyrase (Martin &
Barsotti, 1994; He et al. 1997) to remove ADP contamination in the
fibre and ‘Pé-mop’ (namely 1 mÒ 7-methylguanosine, 0·5 units ml¢
purine nucleoside phosphorylase) to remove Pé contamination
(Brune et al. 1994). Calcium-containing rigor solution was identical
to calcium-free rigor except that the free calcium concentration was
32 ìÒ achieved by addition of Ca-EGTA and there was no apyrase
and no Pé-mop. Loading solution contained 60 mÒ TES, 10 mÒ
EGTA, 1 mÒ free Mg¥, 40 mÒ glutathione, 32 ìÒ free Ca¥,
1·2 mÒ MDCC-PBP, 5 mÒ NPE-caged ATP (pre-treated with
10 units ml¢ apyrase to remove ADP contamination: the final
apyrase concentration in the loading solution was 0·0013 units ml¢),
10 mÒ creatine phosphate, 4 mg ml¢ creatine kinase obtained
from chicken breast (338 units mg¢ at pH 7·1 and 25 °C; Bershitsky
et al. 1996) and Pé-mop.
Protocol
Muscle fibres were transferred from the dissecting stage to the
apparatus by means of a small glass rod and the fibre and T-clips
were attached to the tension transducer and motor hooks in a
trough containing relaxing solution. The temperature of the
microscope stage was adjusted to 12 °C and all subsequent steps
and measurements were carried out at this temperature. Sarcomere
length was adjusted to 2·7 ìm and the width and depth of the fibre
were measured whilst immersed in relaxing solution using the
water-immersion objective lens (Blinks, 1965). The regions of the
fibre which had been exposed to glutaraldehyde during the fixation
procedure could be seen as they were less iridescent than the nonfixed
central region. The length of the central region was measured
as it is needed for calculation of the length change and shortening
velocity. On average the central region prior to shortening had a
length of 2·21 mm (s.d. = 0·25 for 16 fibres), with a range of 1·96 to
3·00 mm. At this temperature and sarcomere length, the isometric
force level reached during activation (190 kN m¦Â, s.d. =40kNm¦Â,
n= 41), was 17% lower than at 2·4 ìm and 15°C (He et al. 1998b)
and resulted in less degradation of the sarcomere signal. The
ATPase activity was also slower, thus allowing more time before
saturation of the MDCC-PBP with Pé. At this sarcomere length, the
percentage of myosin heads in the overlap zone between the thick
and thin filaments is 93 % of maximal, assuming a linear
force—length relationship giving 100 and 0 % overlap at sarcomere
lengths of 2·6 and 4·0 ìm, respectively, as is the case for rat fastand
slow-twitch muscles (Page & Huxley, 1963; Stephenson &
Williams, 1982). Each fibre was incubated for 30 min in relaxing
solution containing 1 % (vÏv) Triton X-100 (He et al. 1998b) to
remove membrane and membrane protein remnants and to improve
its transparency. Fibres were washed twice in relaxing solution and
the sarcomere length was checked again. The sarcomere diffraction
signal was optimized by fine alignment of the He—Ne laser and a
sarcomere length calibration was obtained as described above. The
fibre was transferred to a trough containing calcium-free rigor
solution for 10 min in which rigor tension developed. The fibre was
transferred for 5 min into calcium-containing rigor solution and
then for 7—10 min to a trough containing loading solution. Finally