Properties of skeletal muscle

Figures

Background

Skeletal muscles are the human body’s mechanical output devices.  This tissue converts chemical energy in high-energy phosphate bonds into the mechanical energy of contraction.  Skeletal muscle is striated, highly organized into parallel rows of myofibrils composed of the contractile proteins myosin and actin.  Contraction depends on the presence of ATP, Ca++, and Mg++.  Somatic motor neurons stimulate the muscle fibers, which generate action potentials just like neurons. In skeletal muscle, however, the action potential is coupled to the mechanical event of contraction.  In the body, a motor neuron may innervate 1-1000 muscle cells in a functional group called a motor unit.  Muscle contractions are graded in size by wave summation, which depends on stimulus frequency, or by recruitment of more motor units.

Using a preparation of a fast-twitch vertebrate muscle, the frog gastrocnemius, we will use a semi-isometric recording technique to measure muscle contraction speed and strength.  In isometric recordings, the muscle is kept at a fixed length and tension is measured; isotonic contraction measurements keep the muscle at a constant tension and measure shortening.

Experimental apparatus and tissue preparation

Dissection.  Double pith a 3-4" Northern Grass Frog (Rana pipiens). Cut the skin all the way around the abdomen, and peel away the skin over the legs by pulling down with a pair of tissue-gripping forceps. Sever one leg by cutting through the femur. Remove the muscles of the upper leg and trim the femur to about one inch. Now separate the gastrocnemius muscle from other muscles of the calf, and cut through the Achilles tendon above the ankle leaving most of the tendon attached to the muscle preparation. Next, cut the tibiofibula at the knee, along with the other calf muscles. You should be left with the gastrocnemius and the Achilles tendon at the distal end, and an inch of femur attached to the proximal end of the muscle. Place the muscle in a bath of room temperature Ringer's solution at pH 7.2-7.4.

The muscle will contract in vitro when stimulated by an adequate electrical stimulus, just as it would in vivo when a motor unit is stimulated by a motor neuron.

Grass Stimulator.  The SD9 stimulators are electronic devices that supply a known stimulus to the preparation.  We can set the intensity (voltage), duration (time in msec), and frequency (number of stimulation pulses/sec) of stimulation with the dials on the front panel of the instrument.  Note that the switch below each dial is a multiplier that multiplies the dial value by 0.01 to 10X.

Transducer.  The device on your ring stand is a semi-isometric strain gage transducer (UFI Inc. Model 1030) which converts the mechanical tension produced by muscle contraction into an electronic signal that the recording system can detect.  Other kinds of transducers exist for pressure, temperature, muscle shortening, fluid flow, sound, and light--each of these forms of energy can be converted by a transducer to an electrical signal.

Bridge amplifier and MacLab.  The MacLab units contain 2 to 4 banks of analog-to-digital conversion circuits which will record electrical signals from our preparation.  The bridge amplifier is a Wheatstone bridge circuit which collects data from the strain gauge transducer and provides a signal to MacLab proportional to tension produced by contracting muscle.  In some of the experimental stations the transducer may be connected directly to the MacLab unit (the amplification is built into the MacLab unit)

Connecting the strain gage transducer to the MacLab via a Gould physiograph.  If we are short of bridge amplifiers, one or two groups may have to couple the strain gage transducer to the MacLab unit via a Gould physiograph.  Plug the transducer output into the input of a universal amplifier channel on the Gould physiograph. The amplifier should be set on DC offset, full scale = 10 mV, low pass = 1K, high pass = 100K. Set the chart drive of the physiograph so that paper will run under the pens at about 1-5 mm/sec at first.

Center the pen with either the centering buttons (knob on the Gould 8000 recorder) or with the amplifier offset knob. Check the operation of the transducer by gently pushing on the transducer lever. The pen should deflect "up" with a downward push indicating muscle contraction. Turn the transducer over if this is not the case and make a note on the chart of the direction of pen movement that indicates increased muscle tension.

Check the operation of the signal marker by manually depressing the event button. Plug in the stimulator event marker output to the physiograph's event marker input so that signal pulse is triggered when the stimulator mode switch is activated.

Plug in the physiograph amplifier MON OUT to the MacLab INPUT 1+ via a BNC shielded cable. Plug the stimulator output directly into the MacLab INPUT 2+ via a double-banana to BNC or alligator-BNC cable. [NOTE: do not exceed 10 V stimulus voltages when connected to MacLab or you'll blow the A/D board, a bad mistake that requires expensive repairs.]

Macintosh Microcomputer.  At your table is a Macintosh, a desktop microcomputer that can be used to compose graphs, analyze experimental results, work with text, and record experimental data. You will record data using Chart 3.6.3 MacLab software.  This software simulates a pen-and-paper chart recorder.

Getting started

1. Starting up and working with MacLab.

a.  Make sure the MacLab unit in your station is turned on  -- to turn it on use the power switch on the back of the unit.

b.  Turn on the computer by pressing the power key on the keyboard (to right of "scroll lock").  Allow the computer to power up.

c.  From the apple menu launch Chart 3.6.3., an application that simulates a pen-and-paper chart recorder.

d.  The screen should look similar to Figure 1.  The window shown in Figure 1 looks rather complex, but the software is very user friendly.  We will introduce you to a few key features and you will gradually pick up other details as you work with the program.

  i.  By selecting the Rate pop-up menu you can choose the sampling rate -- the number of data samples that are stored per unit time, and the time taken by each sampling division to scroll past.  For details see Figure 2 below.  By altering the value in this pop-up menu you are doing the same as controlling paper speed in an old-time pen-and-paper recorder.

ii.  By selecting the Range pop-up menu above each channel icon (Figure 3) you can change the sensitivity of your recording.  The sensitivity can be set from 2 mV to 10 V -- the higher the value the less sensitive the recording.  For example, a channel set at a sensitivity of 500 mV will have lower sensitivity than if set at 100 mV.

iii.  At times throughout the experiment and data analysis procedure you will be asked to bring up the Channel Function pop-up menu (Figure 4).  This will allow you to turn the channel off and access settings for the bridge amp you have connected to the MacLab unit, among other features.  Your instructor will demonstrate these features in more detail when appropriate.

iv.  Chart uses a visual metaphor of a mechanical chart recorder:  recorded data scroll across the data display area from the right of the window as if the display area were a roll of paper in such a device, with new data being drawn at the right and old data moving left (see Figure 1). 

To start sampling, simply click the Start button at the bottom right of the Chart window (see Figure 1). -- this means that you will see data be acquired as it moves across the screen.  Whether the data is recorded or not depends on the status of the Record/Monitor icon to the left of the Start button (Figure 1 and Figure 5).  If you place a large X over the Record/Monitor button by clicking it, the data will be displayed but not recorded -- this is a great feature to preview the results of a procedure without using memory to store data.  If the large X is removed from the Record/Monitor icon by clicking on it, the data will be displayed and recorded -- do this only when you are ready to record results of a specific procedure.  For more details see Figure 5.  

v.  Figure 6 shows the changes in appearance that occur at the bottom of the Chart window when you start sampling and in this case also recording.  You can enter a comment while recording so you know what procedure or what settings the experiment involves.  To enter a comment while recording, type in the text entry area at the bottom of the Chart window, and press the Enter or Return key to add the comment to the file at the time the key is pressed.  This features allows you to identify the specifics of an experiment or recording when you scroll through it later. 

To look at several comments at once, locate a comment in a file, or delete or edit comments, choose Comments from the Windows menu to bring up the Comments window (Figure 7).  The comments are listed in a scrolling field in the window in the order that they appear left to right across a file, with the comment numbers in boxes. 

Try out some of the above features of CHART  -- don’t hesitate to try anything! When you’re done exploring, check with an instructor to see that your MacLab is ready to record.

2. Secure the muscle to the apparatus (Figure 8) 

a.  Remove the muscle from its Ringer’s bath.  Attach the muscle to the femur clamp with the bone attached parallel to the clamp and at right angles to the muscle. 

b.  At the top end of the muscle, attach the preparation to the isometric transducer using the fishhook thrust through the Achilles' tendon and tied to the transducer lever.  Use two leaves of the transducer for best sensitivity and isometric recording. 

c.  Attach wires from the stimulator output to the femur clamp (via a screw terminal if possible) and to the fishhook at the top (via a soldered connection).  Polarity (which one is + or -) doesn't matter. 

d.  Once you've got the muscle in place move the clamps apart so as to exert a slight tension on the resting muscle.

e.  Liberally douse the muscle with Ringer's saline solution now and throughout the procedure.  Place a beaker underneath to catch the excess.  It is extremely important that you keep the muscle wet throughout the procedure.

f.  In some units the transducer's output cable plugs into the bridge amplifier which in turn plugs into MacLab Channel 1+ by a BNC shielded cable.  In other units the transducer's output cable plugs directly into MacLab Channel 1. Some lgroups will use a Gould physiograph to couple to transducer to the MacLab unit.  See Getting Started, 1 above for details.

  3. Calibration. 

During the experiments, after the muscle is set in place and between individual procedures, you will calibrate the transducer/MacLab apparatus by placing a weight (10 g) on the end of the transducer lever and recording the displacement caused by that weight.  Record at a moderate speed of 1 - 5  s/div, use Rate/Time pop-up menu to set this. You will use this calibration to compute the tension developed in your muscle under various conditions. 

Repeat the calibration procedure at frequent intervals during your work, and record the new calibration on the MacLab record as a comment or in your notebook for reference.

During experimentation, adjust sensitivity (use Range pop-up menu) as needed (except during an experimental procedure) to yield a good-sized contraction record. 

Always record the settings (especially sensitivity) in your written notes and/or as a comment in CHART.

Liberally douse the muscle with Ringer's saline solution now and throughout the procedure.  Place a beaker underneath to catch the excess.

Experimental procedures

1. Threshold and recruitment of fibers.  For the first experiment, we will find the lowest stimulus voltage needed to start a muscle action potential and cause contraction.

a.  The sensitivity should be set relatively low.

b.  Make sure that the stimulator wires are properly attached to the muscle prep, and the stimulator is set in the following fashion:

Duration:  30 msec

Intensity:  0.1 V

Frequency:  single pulse

c.  Stimulate the muscle with single pulses using the mode switch on the stimulator.

d.  Record at a slow chart speed and note in the Comments the voltage applied for each stimulus. 

e.  Gradually increase the stimulus intensity in 0.1 V or 0.2 V increments, giving a single pulse at each voltage -- keep increasing the voltage until you get a response.  That stimulus intensity is the threshold for the most sensitive fibers in your muscle. 

f.  Continue increasing the stimulus intensity ( now in 0.5 -1.0 V increments) until a maximal contraction is obtained.

g.  Now, with CHART running at a slow/moderate speed, quickly repeat this stepwise progression (0.5 - 1.0 V increments) from threshold to high stimulus intensities to obtain a clean, short record of the effects of recruitment of muscle fibers.  Record comments that include the stimulus voltage used.

h.  Save your data by click-dragging from the File menu down to SAVE or SAVE AS.  Give your file a new name that you’ll recognize and remember.

i.  Investigate the effects of applying a train of repeated subliminal or subthreshold stimuli. Do successive stimuli add together to cause a contraction?

j.  Does duration of the stimulus alter the threshold? Adjust duration to various values and record the voltage needed at each duration to elicit a certain level of response.

2. The muscle twitch. The individual muscle contractions seen with a single stimulation are called twitch contractions.  A maximal twitch involves all the muscle's fibers contracting at once and was observed above at the point when further increases in stimulus voltage didn't cause any further increase in tension.

During this procedure you will record a single maximal twitch at very high speed in Channel 1, also recording in Channel 2 the exact time when you applied the stimulus. 

a.  Calibrate your prep as described above.

b.  Click Channel 2 Range pop-up menu and turn Channel 2 on. 

c.  Use a black probe or dual-banana plug cable provided by your instructor to connect the stimulator output to MacLab Input 2.  NOTE:  Do not exceed 10 V stimulus voltages when connected to MacLab or you'll blow the A/D board, a very expensive mistake. 

d.  Set up Channel 2 to record on 10 V range.  The size of the stimulus wave is irrelevant; all we need is a mark time for the stimulation.  Check with the Input amplifier in the Channel 2 pop-up menu to see that the stimulator pulse is displayed. 

e.  Record several individual twitches at high speed, about 100 - 200 msec/division -- use a maximal stimulus intensity and keep frequency and stimulus duration set as in 1.

Before you start, check that the sampling rate is maximal (400 samples/div) so that you'll be able to precisely measure time in your recordings. You should see the muscle twitch in Channel 1 and the stimulus wave on Channel 2.

f.  Don't forget to bathe the muscle periodically with Ringer's saline solution to rinse away waste products and keep the prep moist.

g.  Save your data again by choosing SAVE from the File Menu. 

h.  Disconnect the stimulator from MacLab Channel 2 and turn Channel 2 off using the Channel 2 pop-up menu. 

3. Twitch fusion and tetany. 

a.  Calibrate your prep as described above.

b.  Use a stimulus voltage which will produce a maximal contraction to investigate the effects of applying a series of repeated stimuli to the muscle. 

c.  Set the stimulator frequency to 1/sec, then 2/sec, then 3/sec, 5/sec, 7/sec, 10/sec, 15/sec, 20/sec.  Stimulus duration stays as above in 1 and 2. 

d.  Apply a train of repeated stimuli by briefly switching the stimulator Mode switch to Repeat or Multiple.  Use only a short period of stimulation, about 5 sec, and be consistent in applying the train of stimuli.  Allow the muscle to rest for about 30 sec. between stimulations. 

e.  Record all information in your notebook--stimulus frequency, voltage, recording speed, sensitivity  -- also insert appropriate Comments during the recording.

The muscle twitches you observe will fuse together, and at high frequency stimulation will reach a condition of tetanic, or constant contraction.  If the recording goes off scale you’ll have to reduce the sensitivity, recalibrate, and repeat the experiment for all stimulation frequencies.

f.   Save your data again by choosing SAVE from the File Menu.

4. Length-tension curve.  You will examine the effects of changing the initial length of the muscle on the tension generated by stimulation with a maximal stimulus.

a.  Calibrate your prep as described above.

b.  Move the transducer up or down and measure the distance between clamps (“muscle length”) with a ruler or calipers.

c.  At each length stimulate the muscle with a single pulse using a maximal stimulus to elicit the maximum muscle tension.  Use only a short range of lengths in your experiment -- perhaps + 0.5 – 1.0 cm total.  Collect 8-10 data points.  For each contraction, save the length value in a comment.  Make sure you recalibrate every time you change muscle length.

d.   Save your data again by choosing SAVE from the File Menu.

5.  Loading.  Examine the effects of loading on muscle contraction.

a.  Invert the apparatus by switching the positions of the femur clamp and transducer.

b.  Hang a pan for weights from the bottom of the muscle, below the transducer, and gradually increase the load by adding 5 to 500 g if your muscle can lift that much.

c.  Remember to recalibrate the transducer.

d.  Examine the effects of after-loading by supporting the transducer lever with a ring clamp. This procedure allows the muscle to start a contraction but as soon as the lever is lifted above the ring clamp load is applied.

6. Fatigue.  When you have completed all the above experiments [not before!], look at the effects of a continuous, high frequency stimulation on muscle tension.

a.  Calibrate your prep as described above.

b.  Reduce the sampling rate and recording time base so you can record over a longer period of time.

c.  Apply a tetanizing pulse (20-50 pulses/sec) and watch the development of fatigue in the muscle.  Record for about 3 minutes, then turn off the stimulator and allow the muscle to relax completely before you stop the recording. 

d.  During the stimulation apply several drops of Ringer's after tension has declined significantly.  Does tension increase after a Ringer's wash?

e.   Save your data again by choosing SAVE from the File Menu.

Clean up

You have completed your experimental procedures.  Before leaving do the following: 

1.  Remove the muscle from the apparatus and dispose of it in an animal waste bag.

2.  Clean all instruments and equipment.

3.  Close the Chart program, shutdown the Mac, and turn off the power in the back of the MacLab unit.

4.  Data analysis will be done next cycle.