Contractile Properties

The proximal end of the EDL muscle was tied to a micromanipulator. The distal end

was attached directly to a sensitive isometric force transducer (Research Grade 60-2999,

Harvard Apparatus, South Natich, MA), thus mimicking the direction of force

development of the muscle in vivo. The muscle was flanked by field-stimulating

platinum plate electrodes attached to a stimulator (Grass S11 stimulator, Quincy, MA)

coupled to an amplifier (CE-1000, Crown Instruments, Elkhart, IN, USA; see figure

3.8). Deflection of the transducer (which was previously calibrated with a calibration

weight of known mass) with a muscle contraction produces a measurable electric signal,

proportional to the force produced. Electrical signals were converted to digital signal by

Powerlab4510 (ADIstruments, Castle Hill, NSW, Australia) running Chart V5.0.2 for

Figure 3.6. Horizontal custom-built plexiglass bath: apparatus used for contractile testing of muscle function

Figure 3.7. The field-stimulating platinum plate electrodes used to stimulate the muscle during contractile testing.

A

B

Figure 3.8. The field-stimulating platinum plate electrodes are attached to a (A) imulator (Grass S11 stimulator, Quincy, MA) coupled to an (B) amplifier (CE-1000, Crown Instruments, Elkhart, IN, USA).

fine adjustments in muscle length (i.e. slowly

e muscle is functioning properly. st

Stimulation of muscle contraction was produced using supramaximal square wave

electrical pulses of 0.2msec duration. Optimum contractile length (Lo) of the muscle

(the length at which maximal contractile force is produced) was established with a

series of twitch contractions, and

stretched out). This ensures maximal activation of each muscle was attained and thus

comparisons between muscles could be made. Muscle length at Lo was measured with a

vernier caliper at the point at which the muscle is connected to its tendons.

Having established Lo, single muscle stimulations (eliciting muscle twitches) were

elicited at 40V and increased by 2 volts, until a plateau in twitch force (Pt) was reached

(optimal voltage). The idea of these stimulations was to stretch the series elastic

elements of the muscle, therefore allowing a good release of calcium and to ensure that

A single tetanic stimulation at 100 Hz was performed, with three minutes recovery,

followed by a single twitch. The tetanic stimulation is performed to test that the knots

are tied correctly, before subsequent experiments can be continued. The single twitch

performed at the end of 3 minutes recovery enables us to observe if there is any post-

tetanic potentiation, due to sustained phosphorylation of light chains on the myosin

heads from the previous tetanic stimulation. This was evident by an increase in the Pt

that was previously observed. A lower Pt indicated that the knots had slipped. In this

case, the muscle is lengthened out slowly again, until optimal length was re-established.

(Po). Single tetanic contractions were stimulated

over frequencies ranging from 10Hz to 140Hz, with 3 minutes recovery in-between

contractions (see figure 3.9 and 3.10). Optimal stimulus frequency and maximum

teta en complete fused tetanus occurred. The force

eveloped at each frequency was expressed relative to the peak force obtained. The

optimal length, voltage and stimulus frequency established was used for the remainder

of the experiments.

Three Pt measurements were performed. For each of these twitch contractions, the

following measurements were made;

¾ Peak Force (Pt)

¾ Time to Peak Tension (TTP)

¾ Half Relaxation Time (1/2 RT)

Force-frequency measurements were used to establish optimal stimulus frequency and

maximum isometric tetanic tension

nic force (Po) was established wh

Time to peak tension (TTP) is an unrefined indicator of the Ca2+ release process, the

binding to troponin-C and the activation of actin and myosin. Half-relaxation time

(½RT) is an approximate measure of the ability of the sarcoplasmic reticulum Ca2+-

ATPase to re-sequester Ca2+ (see figure 3.9 & 3.10).

Following contractile testing, the muscle was removed from the bath. Under a

microscope (Lerica, ZOOM 2000TM Z45V, China) surgical silk loops and other debris

(including proximal and distal tendons) were removed. The muscle was then blotted dry

on filter paper and weighed on an analytical balance (OHAUS, GalaxyTM 160D, Ohaus

Corporation, NJ, USA). The muscle was then divided into two sections; one half was

snap-frozen in isopentane, cooled in liquid nitrogen. The muscle was placed in a

labelled cryule, and stored in liquid nitrogen (–172oC), for later determination of fibre

size and proportions, and percentage of damaged area (rate of regeneration). The other

portion was immediately snap frozen and stored in liquid nitrogen (–172oC) for later

assessment of muscle metabolites and protein analysis. The same procedures were

performed on damaged soleus muscle and the contralateral non-damaged EDL and

soleus muscles.

Following removal of all muscles required for analysis, rats were killed by overdose of

anaesthetic. Peak tetanic forces were expressed relative to the muscles cross-sectional

area (specific force-sPo) enabling comparisons between muscles of different area and

length. Cross-sectional area (X-A) was calculated by dividing MM (muscle mass) by

(fibre-length x density), where fibre length is obtained from the FL/ML (fibre length:

muscle length) ratio, which is 0.44 in the EDL and 0.71 in the soleus (Brooks and

Faulkner, 1988) and density equals 1.06 g/cm3 (Close, 1972). The degeneration and

fibers grow within the preexisting basal laminae, and therefore the angle of muscle fiber

insertion is not altered (Gregorevic et al., 2002). Specific force was calculated by

dividing Po by the X-A (refer to Appendix B for example calculation).

TTP ½ RT Pt Po @ 10Hz Po @ 25Hz Po @ 75Hz Po @ 130Hz (Nm) 0 1000 2000 3000 4000

Figure 3.9. A typical example of an EDL twitch contraction (Pt) showing how TTP and ½ RT was measured. TTP = time from the start of the contraction to peak tension; ½ RT

= half relaxation time from peak tension. Single tetanic contractions (Po) were

stimulated over frequencies ranging from 10Hz to 130Hz.

(Nm)) 0 500 1000 1500 2000 TTP Pt Po @ 10HZ Po @ 25HZ Po @ 75HZ Po @ 130HZ ½ RT

½ RT = half relaxation time from peak tension. Single tetanic contractions (Po) were 10Hz to 130Hz.

Figure 3.10. A typical example of a soleus twitch contraction (Pt) showing how TTP and ½ RT was measured. TTP = time from the start of the contraction to peak tension;