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SKELETAL MUSCLE
PURPOSE:
This laboratory exercise uses stimulus-response experiments to explore some of the functional properties of skeletal muscle.
THEORY/INTRODUCTION:
Certain proteins known as "contractile proteins" (i.e. actin and myosin) are the basis for force and movement in living cells. Contractile proteins can convert chemical energy such as that stored in ATP (adenosine-5’-triphosphate to mechanical energy of motion.
Muscle cells represent structures having concentrations of contractile proteins. There are three types of muscle cells: skeletal, smooth, and cardiac. Skeletal muscle, as the name implies, is connected to the bones of the skeleton and causes their movement. Smooth muscle is associated with hollow organs such as intestine, stomach, uterus, blood vessels (i.e. veins and arteries), and urinary bladder. Cardiac muscle forms the bulk of the heart. Skeletal muscle is the most massive tissue of the body. In humans, it represents about 40-45% of the total body weight. Individual skeletal muscle cells are long and cylindrical in shape, with a diameter ranging from 10-100 microns. Single cells are called "muscle fibers" (i.e. "myofibrils" or "myocytes"). The word "muscle" refers to a bundle of fibers.
MUSCLE CONTRACTION
Muscle contractions are triggered by nerve impulses conducted via nerve fibers to the neuromuscular junction (i.e. motor end-plate). Upon reaching the end of the axon, a nerve impulse (action potential) induces the release of a quantum of acetylcholine vesicles which diffuse across the cleft (i.e. synapse) between the motor end-plate and muscle fiber. The acetylcholine reaction causes rapid depolarization of the muscle cell membrane. A short time later, acetylcholinesterase, an enzyme present in the motor end-plate, neutralizes the acetylcholine causing membrane repolarization back to the resting state. Each action potential initiated at the neuromuscular junction propagates (i.e. moves) along the muscle fiber, bringing about a single contractile twitch.
Muscle fibers are organized functionally in groups. The muscle fibers (i.e. "myofibrils"), motor end-plate, and the neuron which excites it is called a "motor unit" (see below).
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Experimentally, it is possible to cause a muscle to twitch by shocking it electrically with a pulse of current. Direct electrical stimulation of the muscle will be used in the chapter to demonstrate some of the physical properties of skeletal muscle.
The two classes of muscle contractions are "isometric" and "isotonic". A muscle contracting under isometric conditions generates tension but does not change length. In the body, an example of muscles contracting isometrically would be the trunk (i.e. thoracic region) muscles which maintain posture. "Isotonic contraction" refers to muscle which shortens (i.e. changes its length) under a constant weight load (i.e. no change in tension). There are two types of mechanical transducers, Force and Displacement, available for use in recording "isometric" and "isotonic contractions", respectively. The word "transducer" means "to change" or "to convert". In this instance, a mechanical signal (i.e. muscle action) is converted to an electrical signal.
FROG MUSCLE PREPARATION: (see lab manual on each bench for details)
To save time, several students can proceed directly to study the electronic equipment, while the others surgically prepare the frog muscle.
The TA will supply each lab group with a freshly pithed frog. A frog is pithed by running a needle into the brain and spinal column, effectively destroying the central nervous system. This procedure renders the frog insensitive to stimuli, While the frog is "dead" in the conventional sense, the heart will beat, and the normal metabolic functions will continue for several hours.
Whereas almost any skeletal muscle of the frog could be used, the gastrocnemius muscle (i.e. "calf muscle") is particularly well suited for experimentation because of its size and its prominent Achilles tendon.
The Figure below shows the location of the gastrocnemius muscle in the frog. (See lab manual Figures 4-5 for details.) Both the gastrocnemius muscles can be used in the experiments. Using small scissors, remove all the skin from both legs of the frog. Keep the exposed muscles moistened with Ringer’s solution.
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Dissect away the muscles of the upper leg to expose the femur bone. With heavy shears, cut the femur as close to the hip joint as possible. Tie fish-line around the Achilles tendon and then snip the Achilles tendon from the end of the heel. Proceed to free the gastrocnemius muscle from the tibia. Cut the tibia off below the knee. A schematic of the completed muscle preparation is shown below.
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MUSCLE TWITCH CHARACTERISTICS: (you can use either displacement or force transducer - each must be used at least once
This section examines the characteristics of the muscle twitch (i.e. single contraction). A twitch is the contraction response a muscle shows when excited by a single stimulus of short duration. (see below).
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USING THE DISPLACEMENT TRANSDUCER (most sensitive)
The gastrocnemius muscle preparation attached to the Displacement Transducer is illustrated in below. Tighten the Femur Clamp around the femur bone close to the knee joint. Attach the fish-line between the tendon and the muscle lever. The length of the fish-line between the tendon and the muscle lever should not exceed 4 cm. Set up the weight hanger with a 10 gram (gm) weight. Move either the right angle clamp holding the Femur Clamp or the Displacement Transducer clamp until the muscle lever is almost horizontal. Adjust the loading screw to take the tension off the muscle. Position the Stimulating Electrode against the middle of the muscle (i.e. belly). Remember to moisten the muscle frequently with Ringer’s solution. The calibration for the Displacement Transducer as shown below is: 1 mm change in muscle length = 100 millivolts (mV) change at the "output" of the Type 400 Bioamplifier Supply.
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USING THE FORCE TRANSDUCER
When using the Force Transducer, keep in mind that the muscle will contract under isometric conditions (i.e. no change in length). Hence, the recording of the muscle twitch is tension versus time. The figure below shows the Force Transducer set-up. Tighten the Femur Clamp around the femur bone close to the knee joint. Pass the fishline through the Force Transducer loop and tie a secure knot. Lower the Femur Clamp to apply a slight tension to the muscle. Position the Stimulating Electrode against the middle of the muscle. Moisten the muscle frequently with Ringer’s solution. The calibration for the Force Transducer is: 1 gm change in muscle tension = 20 mV change at the "output" of the Bioamplifier Supply.
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MUSCLE TWITCH
Use the zero adjust controls ("coarse" and "fine") on the Strain Gage Preamplifier to adjust the position of the Strip Chart Recorder pen. Set the Stimulator to deliver single pulses 10 milliseconds (ms) in duration and 10 volts in amplitude.
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USING THE CHART RECORDER
Stimulate the muscle with single shocks while recording the displacement. If necessary, readjust the vertical sensitivity of the Strip Chart Recorder from 200 mV/cm to provide adequate deflection for your particular set-up. Measure the latency period, contraction interval, and relaxation interval of the muscle twitch. The stimulator can be connected to the other channel on the strip chart to give a deflection when the muscle is stimulated. This mark allows for easy measurement of the latency period. The beginning of the rising edge of the trace left by the stimulation marks the advent of the stimulus applied to the muscle. To measure these intervals, use a fast paper speed.
THRESHOLD & GRADED RESPONSE CHARACTERISTICS:
This section demonstrates the graded response characteristics of a muscle.
The stimulus-response for a single muscle fiber is "all or none". A bundle of muscle fibers does not behave in this fashion. The graded response is due to the fact that not all of the muscle fibers in a bundle will experience that same local stimulus magnitude for a given stimulus. In addition, not all muscle cells have the same threshold.
Either the Displacement or Force Transducer can be used for this experiment. Position the Stimulating Electrode against the middle of the muscle. Set the Stimulator to deliver single pulses of 10 ms duration. Start with the pulse magnitude at zero. Gradually increase the amplitude until the response of the muscle is just detectable. This is the "threshold stimulus". Continue to stimulate the muscle with single pulses increasing in magnitude until a point is reached (maximal) where any further increase in the magnitude of stimulation does not increase the magnitude of the response. Plot the results (voltage vs. response)
WORK DONE BY SKELETAL MUSCLE:
This section determines the ability of the muscle to do work. By using the Displacement Transducer, it is possible to measure accurately the length of contraction of the frog gastrocnemius muscle. With the muscle weight attached to the hole farthest (5 cm) from the pivot on the muscle lever, the "output" of the Bioamplifier Supply is 100 mV/mm of displacement.
Position the Stimulating Electrode against the middle of the muscle. Set the Stimulator to deliver single pulses 10 ms in duration and 10 volts in amplitude (maximal stimuli). Keep the muscle moist with Ringer’s solution.
Begin by stimulating the muscle with a 10 gm weight in the weight hanger. Continue to increase the weight load in increments until the muscle fails to lift the load. Record the muscle displacement for each value of the weight load.
The actual work load performed by a muscle is given by:
W = M g D
where: W = work in joules
For this experiment, the work can be expressed as:
W = M D
where: W = grams x centimeters
Plot the values of W found for the frog gastrocnemius muscle at different load values.
SUMMATION EFFECTS IN SKELETAL MUSCLE:
This section demonstrates the "summation" phenomenon in skeletal muscle. In the previous sections of this laboratory, the frog muscle was stimulated only by single pulses, causing a single twitch/pulse. Skeletal muscle has a relatively short "refractory period" (the period during which the muscle cannot be excited by a second stimuli). This means that multiple stimuli occurring at close intervals will lead to multiple contractions. As the frequency of stimulating pulses increases, the muscle has virtually no time for relaxation. A phenomenon known as "summation" occurs where the effects due to each stimulus add up to produce a higher tension or displacement than would result from just a single stimulus (see lecture notes and textbook).
Either the Displacement Transducer of Force Transducer can be used to demonstrate summation effects. Set the Stimulator to deliver continuous pulses 10 ms in duration and 10 volts in amplitude. Begin stimulating the muscle at a frequency of about two pulses/sec. Gradually increase the frequency of the pulses while observing the recording of the muscle contraction response. Notice that the amplitude of the contraction reaches a point known as "fused" or complete "tetany", where the muscle remains in a steady state of contraction. The muscle is said to be "tetanized".
Turn off the Stimulator after tentanizing the muscle for a few seconds. Observe the relatively long relaxation period.
Remember to have the strip chart recorder set in the DC position.
LENGTH-TENSION RELATIONSHIP:
This section demonstrates that the maximum tension a muscle can develop depends on its final length.
A sketch of the isometric length-tension curve of skeletal muscle was given in class and in your textbook.
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The most striking feature of the curve is that maximum tension occurs at length l0, the "resting length". In fact, the "resting length" of a muscle is defined as the length at which the greatest possible contractile tension is developed. In the body, the skeletal muscles normally have the "resting length". The length-tension relationship of skeletal muscle supports the Sliding Filament Hypothesis of H.E. Huxley and A.F. Huxley. Briefly, the Sliding Filament Hypothesis of muscle contraction states that muscle contraction results from the relative movement of "thick" and "thin" filaments past each other, rather than from a reduction in length of the filaments. At the "resting length", there is a maximum interaction between the "thick" and "thin" filaments (cross bridges), hence, greatest tension is possible. At lengths longer or shorter than the "resting length", the "thick-thin" filament interaction is less, and therefore the maximum tension developed is also less.
It is possible to demonstrate experimentally the length-tension relationship with the frog gastrocnemius muscle. Use either the displacement transducer (change the loading screw and make measurements with a ruler) or the force transducer (physically move the transducer and measure with the ruler). These are very course measurements and you can more closely examine this phenomenon using the computer model.
Finish the laboratory using the computer model (see handout).
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