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 F_max. 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 V_max. 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 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

• 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, 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 choose the maximum 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.” Under “Trigger", set “Delay” to zero ms (note - you may not be able to do this until you create a stimulus event).  With this setup, the time when the stimulus is given to the muscle will be considered t = 0.

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.
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.

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.

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 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 F_max 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 F_max.
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.

• 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 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.