Biology 450 - Animal Physiology Lab
Fall 2007
Lab 6 – Muscle Contractile Kinetics
In this lab, you will measure the contractile kinetics (variables related to force
production and shortening velocity) for a model muscle, the gastrocnemius muscle
of the bullfrog, under in vitro conditions.
Background
You learned in lecture about various aspects of muscular contraction and the
conditions under which contraction can be measured. Stimulation of muscles under
isometric (fixed-length) and isotonic (fixed-load) conditions allows measurement of
force production and shortening, respectively, and variation in the stimulus
delivered to the muscle allows comparison of twitch (single action potential) and
tetanic (multiple action potential) contractions.
Measurements of muscle twitches under isometric conditions allow determination of
the amount and time course of force development. The maximal force a muscle
develops during a twitch is Fmax. Three variables are typically measured for the time
course of contraction: the latent period (the delay between the action potential and
the start of force production), the contraction time (the time from the start of force
production to peak force), and the 50% relaxation time (the time from peak force
until force production is 50% of peak). The latent period and the rise in force before
peak force production are caused primarily by the time it takes calcium ions to
diffuse from the sarcoplasmic reticulum (SR) into the myofibrils. Similarly, the slow
decline in contraction is a result of the time required for Ca2+ to be resequestered
by the SR and to diffuse out of the myofibrils.
Isometric measurements also make it possible to determine force production as a
function of muscle length. This would be similar to the length-tension curve for a
single sarcomere, discussed in lecture. In general, the force a muscle develops will
depend on the degree of overlap of thick and thin filaments in the muscle, which is
determined by the muscle’s current length (relative to its normal resting length).
Isotonic measurements of muscle contraction can be used to measure muscle
shortening, both as a simple value and as a rate. Both the degree and rate of
shortening depend on the load a muscle is trying to move and on the force the
muscle produces. If a muscle is stimulated to produce force while attached to a load
that is too heavy for it to move, the muscle cannot shorten and its shortening
velocity will be zero (such a contraction becomes de facto an isometric contraction).
Decreasing the load on the muscle will allow the muscle to shorten once it
generates sufficient force to move the load. Smaller loads result in faster
shortening velocities. If there is no mass whatsoever attached to the muscle, the
rate at which it shortens is called its maximum shortening velocity, or Vmax.
Measurements of shortening velocity under different loads can be used to construct
a force-velocity curve, which relates the two variables graphically. Different muscles
differ in the relationship of shortening velocity to force, with these differences
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usually related to the functions of the muscles in the living animal (e.g. your eyelid
muscles can shorten very quickly but many of your postural muscles cannot). You
created a (somewhat crude) force-velocity curve in Lab 5.
While the total force generated during a twitch contraction should depend only on
the muscle’s length, regular stimulation of the muscle at sufficiently high frequency
will produce a tetanic contraction. During a twitch, Ca2+ may not reach all the
myofibrils, and much of the force generated by the crossbridges is absorbed in
stretching the internal non-contractile components of the muscle and does not
appear in our measurement of force. If the muscle is stimulated several times fast
enough so that it cannot relax between stimuli, the total force rises and remains
elevated, as long as the stimuli continue and the muscle does not fatigue. This is
known as a tetanic contraction. The non-contractile components of the muscle are
fully stretched and most of the force generated by the cross bridges can be
measured.
In this lab, you will use the gastrocnemius muscle of a bullfrog (Rana catesbeiana)
to determine the following for a model muscle:

The time course of force production during a twitch

The length-tension curve for the muscle

The force-velocity curve for the muscle

The stimulus frequency required to generate a tetanus

The relative force production during twitch to tetanic contractions.
Lab Procedures
Note:

This lab involves the use of freshly sacrificed animals. Be aware that
some spinal reflexes and the like may still be apparent.

At least one member of your lab group will need a dissecting kit.
In these experiments, you will be measuring force production and positional
information (which can be used to determine speed) using transducers designed for
each function. Your initial goals as a group will be to dissect the muscle out of your
frog and to calibrate your two transducers.
Split your lab group into two. One subgroup will do the muscle dissection, and the
other will set up the muscle rig and calibrate the transducers.
Setup
Hardware
You will use a force transducer and a position transducer, in combination with
Scope, to measure most of the variables of interest in these experiments. You will
put together a muscle rig using a ringstand, ringstand clamps, a tension adjuster,
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and the transducers. Two slightly different configurations will be used for the four
experiments.
1. Start by attaching the tension adjuster to the bottom of the ringstand and
the force transducer to the top. The force transducer has two hooks – you
will be using the one closest to the ringstand. The hook should be directly
above the bar of the tension adjuster.
2. Place a metal tray under the rig to catch the saline you will be applying to
the muscle.
3. Make sure that the bridge amp is plugged in and that the gain for channels 1
and 2 is set to 1´.
4. Connect the plug from the force transducer to the back of the bridge amp
(ETH-400) on channel 1, and the plug from the position transducer into
channel 2.
5. Use BNC cables to connect the output from the amp’s channels 1 and 2 to
input channels 1 and 2 of the PowerLab unit.
6. Connect the stimulus electrode to output 1 on the PowerLab. Leave the
needle guard(s) on until the muscle is in the rig.
Software
You will use Scope to gather data in these exercises. Scope is used rather than
Chart to capture the relatively rapid events associated with a twitch contraction.
1. Start the Scope program and make sure it is set to view two channels.
2. Under “Time Base” on the main panel, set the time to 500 ms as a starting
value, then the number of samples to 2560.
3. Under “Input A,” set the range to 2 V as a starting value, and under “Input
B” select 200 mV.
4. Select “Sampling...” from the Setup menu and make sure that “Sweep Mode”
is set to “Single”. Under “Sweep,” set “Source” to “User.”
Calibration
In order to determine what forces or distances are represented by the voltages
produced by your transducers, you will need to calibrate them. This process
requires a set of weights (for the force transducer) and a ruler (for the position
transducer).
Note:

To avoid a possible electrical short, select .... Stimulator...” from the
Setup menu and set “Mode” to “Off.”
To calibrate the force transducer:
1. Click the “Input Amplifier...” button under Input A in the main window of
Scope.
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2. With no weight on the transducer hook, click the “Display Offset...” button to
bring up the Input Voltage window. Now adjust the input offset knob on the
bridge amp so that the input voltage is as close to zero as possible, then
close this window
3. Put a 5 g weight on the weight caddy (which also weighs 5 g) and hang the
caddy from the 100-g-range hook of the force transducer. You should see a
slight increase in the voltage in the Input Amplifier window. When the caddy
is steady, click the “Units...” button to open the Units Conversion window.
4. In the Units Conversion window, use the mouse to select a portion of the
mini strip chart that shows the voltage when the caddy was steady. Then
click on the right arrow next to the text box in the upper left of the
window. This automatically enters the current voltage reading into the text
box. Then enter the mass (10 g in this case) into the text box to the right to
indicate to the program that the current voltage reading is equivalent to 10
g.
5. The next part is less than straightforward, but is the quickest way to finish
the calibration: Enter a set of dummy values for the second calibration
values - they can be anything other than the numbers for the first set of
values. Click “OK” to exit the Units Conversion window.
6. Repeat steps 3 and 4 but with a total mass of about 100 g, and use the lower
pair of text boxes to enter the values.
7. Use the drop down menu for “Units” to select grams (adding this choice if
needed). When done, click “OK,” then “OK” again to leave the Input
Amplifier window. You may want to adjust the scale on the Y axis.
To calibrate the position transducer:
1. Click the “Input Amplifier...” button under Input B and proceed generally as
above except as indicated.
2. Clamp the transducer in the rack in a vertical orientation, make sure the
sliding rod is all the way down and then set the voltage offset to zero using
the bridge amp.
3. To make the next steps easier, turn the transducer so that the sliding bar is
horizontal (remember which end was up!). Now, move the bar out 5 mm
from its position in 2
4. Obtain the voltage by clicking the right arrow and set the value to 5 mm (you
are only interested in the change in position, so where you define zero is
irrelevant).
5. As for 5 in the last procedure.
6. Move the bar 30 more mm (total distance = 35 mm) and get the second
voltage reading.
7. Select or add “mm” as the units.
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Dissection
You need to dissect free the gastrocnemius (calf) muscle of a frog leg for use in the
muscle rig. You will be supplied with either a whole frog or a frog leg by one of the
instructors.
During the dissection, be sure the muscle does not get too dry. Apply frog Ringer’s
as necessary. It may be helpful to pin down the limb during dissection, but avoid
sticking any pins through the muscle.
1. Make an incision in the skin along the
ventral surface of the leg. Use scissors
rather than a scalpel to avoid cutting too
deeply. Peel back the skin from the leg.
Be careful not to tear any muscle fibers.
2. Locate and cut through the Achilles’
tendon, leaving a piece long enough for
the insertion of a hook.
3. Remove the membrane surrounding the
muscle.
4. Detach the muscle from the leg, starting
from the distal end and working your way
toward the origin (upper attachment
point) of the muscle. It will probably be
necessary to cut through some of the
connective tissue holding the muscle in
place. Do not detach the muscle from its
point of origin. Remove as much of the surrounding tissue from the origin as
possible, until you can clearly see the ligament that attaches the
gastrocnemius to the femur.
5. Cut away as much of the femur as possible so that only a small piece of bone
remains attached to the muscle.
6. Tightly tie a piece of thread about 15 cm long around the ligament. Make
sure the knot will not slip.
7. Measure the length of the muscle and weigh the muscle. Then keep the
muscle in saline until the muscle rig is assembled and calibrated. Bring the
frog’s remains to the instructor for disposal.
8. When the rig is ready, hook the Achilles tendon onto the small wire hook on
the tension adjuster (You can remove the hook from the bar first. It may
help to first punch a hole in the tendon using a sharp probe.) Then tie the
other end of the muscle to the hook on the force transducer using the thread,
adjusting the position of the force transducer and/or the screw on the tension
adjuster to put a very small amount of tension on the muscle.
Note:

Apply frog Ringer’s to the muscle regularly to keep it moist.
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Stimulus Voltage
To make the muscle prep last as long as possible, the minimum stimulus voltage
that achieves a good result should be used. You need to determine what voltage to
apply to get a dependable contraction.
1. Insert the electrode pins through the belly of the muscle, with about 2 cm
between them.
2. Make sure that the muscle is under just enough tension so that there is no
slack in the system. This arrangement prevents the muscle from shortening
and thus makes any contractions isometric.
3. In Scope, open the Stimulator window using the menu item under Setup. Set
“Mode” to “Pulse” (= single), delay to zero, and duration to 10 ms. Set the
voltage range to 10 V for now, then set the stimulus amplitude to 10 V.
Close the Stimulator window.
4. Test the muscle preparation by applying a twitch stimulus (click the “Start”
button on Scope’s main window). You should see the muscle contract or
jump briefly. If nothing happens, try to determine what is wrong with the
setup - ask for assistance if needed.
5. A suitable stimulus voltage for these exercises will produce a twitch force
equivalent to no more than 40 g. To identify this voltage, start at a low
voltage (about 0.1 V) and slowly increase stimulus voltage by 0.1 - 0.5 V
intervals. (Note that the PowerLabs are limited to a 10 V output.) Record
the voltage you decide to use.
Note:

The force produced by the muscle is the maximal force minus the resting (or
baseline) force. The force transducer will typically measure some force even
when the muscle is at rest due to the weight of the muscle plus any tension
on the muscle.

This resting force should not be greater than the equivalent of about 20 g or
so. If it is higher, you should decrease the tension slightly.

Do not move the electrodes after you have selected your stimulus voltage moving the electrodes will likely change which motor units are stimulated,
changing the force production of the muscle.
Exercise 1 - Force generation during twitch
Determine the latent period, contraction time and relaxation time of an isometric
twitch.
1. Using the same setup, record a twitch generated using the stimulus voltage
selected above.
2. Determine maximal force generated (being sure to correct for the baseline
value). Also calculate the latent period (delay between the stimulus and the
onset of force generation, the contraction time (the time between the onset
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of force generation and maximal force) and the 50% relaxation time (the
time from peak force until force production has declined to 50% of peak).
3. Make a printout of your twitch recording.
Exercise 2 - Length-tension relationship
Determine the relationship between muscle length and Fmax by varying the length at
which the muscle is held during isometric twitch contractions.
1. Begin again with minimal tension on the muscle. Measure the length of the
muscle, using clear landmarks (e.g. the location of the hook and thread
loop). This will be considered the normal resting length of the muscle.
2. Generate a twitch contraction and determine the maximal force generated.
3. Increase the length of the muscle by a few mm using the tension adjuster.
4. Generate another twitch contraction and determine the maximal force
generated. Be sure to record the length and Fmax.
5. Repeat steps 3 and 4 until the muscle is about 50% longer than its starting
length, or force production drops to about a third of its original value,
whichever comes first.
Note:

Your muscle prep may fatigue with continued stimulation. You may want to
let it rest between exercises.
Exercise 3 - Force-velocity relationship
Using an isotonic configuration, you will gather data to create a force-velocity curve
for an isolated muscle. Obtaining accurate contraction velocities for light loads is
difficult using our setup (for a variety of reasons), so data in this range may be
somewhat suspect. Consider the results for heavier loads to be more reliable.
1. Remove the tension adjuster and replace it with the position transducer. The
hook from the tension adjuster can either be left in, and the position
transducer hooked to that, or that original hook can be replaced by the one
on the position transducer. Locate the position transducer so that the top
nut of the moving rod just rests against the top of the transducer box. (In
other words, the muscle should not quite by supporting the weight of the
rod.) This will prevent the load you put on the muscle from stretching it.
2. Perform a few experimental twitches to be sure the rod moves freely. Also,
see if you can reduce the voltage range for the position transducer to help
increase the signal to noise ratio. (Since the position transducer moves only
a small amount in most cases, the voltage outputs may be much less than
those seen when the rod moves over its full range.)
3. Attach the weight caddy to the bottom of the force transducer and begin
gathering data on twitches under different loads. If possible, determine the
approximate maximum load the muscle can be loaded with and still shorten.
Note – you may want to mix the order of your weight trials to avoid the
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possibility of fatigue confounding your results. Let the muscle rest for 30
seconds or so and see if this affects the contractions.
4. Determine the maximal shortening distance for each load (i.e., how far the
muscle can raise each load).
5. Determine the contraction velocity for each load by calculating the slope of
the position data (velocity = distance/time). Be consistent in how you select
the portion of the total curve you use for each trial!
6. Print out three representative contractions for a light, medium and heavy
load.
Experiment 4 - Tetanic contractions
Tetanus can be induced by giving the muscle a series of frequent electrical signals.
As stimulus frequency increases, successive twitches begin to meld into one
another. At some stimulus frequency, the twitches become fused and a smooth
trace is obtained.
1. Return the muscle to the isometric configuration (tension adjuster holding
the bottom of the muscle). Remeasure the following muscle parameters for
a (new) single twitch: latent period, contraction time, time to 50%
relaxation, and maximal force.
2. Open the Stimulator window and change the mode to Mode to “Multiple.”
During these trials, you should administer trains of stimuli that last no more
than about a half-second. (You may have adjust the sweep time to see the
entire period of contraction.)
3. Begin by administering a train of stimuli at 1 Hz*, then increase the
frequency of stimuli to 2.5, 5, 10, 15, 20, etc. Hz in each trial until tetanus is
seen. Wait at least thirty seconds between successive trials.
4. Determine the stimulation frequency required before there is any increase in
contraction force (summation), and the frequency required before true
tetanus is observed. When a tetanus is generated, measure latent period,
rise time (time from baseline to maximal force), time to 50% relaxation
(measured from the beginning of relaxation), and maximal force.
5. Make a printout of a tetanus recording.
* Scope does not allow you to set the frequency directly. Instead you set the time
interval between pulses. This is made more complicated by the fact that the
interval does not include the time when the stimulus occurs. Below is a table
giving the appropriate interval for different pulse frequencies, given a pulse
duration of 10 ms.
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To get this
stimulation
frequency
Use this
interval
1 Hz
990 ms
1.5
657
2
490
2.5
390
3
323
4
240
5
190
6
157
8
115
10
90
12
73
15
57
20
40
25
30
30
23
35
19
40
15
45
12
50
10
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