Lab #9: Muscle Physiology

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Lab #9: Muscle Physiology
Background
Sarcomere
Thick Filaments
Overview of Skeletal Muscle Contraction
Skeletal muscle fibers are very large, elongated
cells (Fig 9.1). Roughly 80% of the content of
each muscle fiber consists of long bundles of
protein called myofibrils. The myofibrils, in
turn, consist of two types of myofilament (Fig
9.2). One type of myofilament, called the thick
filament, is composed of hundreds of molecules
of a protein called myosin. The other type of
myofilament, the thin filament, contains three
different proteins: a structural protein called
actin that can form bonds with myosin, a protein
called tropomyosin that regulates binding
between myosin and actin, and the calciumbinding troponin which regulates the position of
tropomyosin.
The two myofilaments are
arranged in the myofibrils in distinctive repeated
structures called sarcomeres. Each sarcomere
contains a series of thin filaments at either end
that partially overlap with thick filaments found
in the center.
Muscle contracts through an ATP-driven
Fig 9.1. A micrograph of segments of skeletal muscle
fibers. N = nucleus, CT = connective tissue, M =
myofibrils. Note the alternating light and dark banding
pattern created by the repeated sarcomeres along the
lengths of the myofibrils. Image is from www.vms.hr
/atlas/ histology/08/ah08202.htm
Thin Filaments
Troponin
Tropomyosin
Actin
Myosin
Fig 9.2. Arrangement of myofilaments into sacromeres
within a myofibril (above) and the structure of thick
and thin filaments, illustrating the proteins that make
up each.
interaction between actin and myosin called
crossbridge cycling (Fig 9.3). First, the globular
head of a myosin molecule extends laterally and
binds with a complementary binding site on an
actin molecule to form a bond called a
crossbridge. Then, in a process called a power
stroke, the globular head bends inward towards
the center of the sarcomere, pulling the thin
filament with it. The crossbridge then breaks,
and the globular head of the myosin unbends,
preparing the myosin molecule to repeat the
process. As a result of many myosin molecules
alternately binding the thin filaments and pulling
them inward, the thin filaments are pulled over
the thick filaments toward the center of the
sarcomere, thus shortening the overall length of
Myosin Head Unbends
Myosin Binds to Actin,
Forming Crossbridge
Myosin Releases Actin,
Breaks Crossbridge
Power Stroke Pulls Thin
Filament over Thick
Fig 9.3. An outline of crossbridge cycling
Lab #9: Muscle Physiology
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Without Calcium – Crossbridges Cannot Form
Fig 9.4. The sliding filament mechanism of muscle
contraction. Myofibrils contract by the thick filaments
pulling the thin towards the center of the sarcomere,
increasing the degree of overlap between the thick and
thin filaments.
each sarcomere and, in turn, the length of the
muscle (Fig. 9.4).
Crossbridge cycling, and hence muscle
contraction, can only occur under specific
conditions. This is because normally troponin
positions tropomyosin on top of myosin-binding
Somatic Motor Neuron
1
2
3
4
Myofibrils
Motor End Plate
Thin filaments
Thick filaments
Transverse Tubule
Sarcolemma
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Sarcoplasmic Reticulum
Fig 9.5. Excitation of a skeletal muscle fiber. The
sarcoplasmic reticulum has been removed from the left
side of the illustration to show the arrangement of the
thick (myosin) and thin (actin) filaments in the
sarcomeres of the myofibrils.
Skeletal muscle
excitation typically occurs in the following series of
events enumerated in the illustration: 1) The binding of
acetylcholine from a somatic motor neuron to
chemically gated ion channels on the motor end plate
(subsynaptic membrane) triggers an action potential in
the sarcolemma. 2) The action potential propagates
down the length of the muscle fiber. 3) When the
action potential reaches the openings of transverse
tubules, the depolarization is conducted down these
tubules and into the interior of the cell. 4) The
depolarization of the transverse tubules induces the
opening of Ca2+ ion channels in the sarcoplasmic
reticulum, and Ca2+ is released into the cytosol.
With Calcium – Crossbridges Form
Fig 9.6. Ca2+ triggers skeletal muscle contraction. In
the absence of Ca2+, troponin positions tropomyosin on
the thin filament in such a way that it blocks myosin’s
globular heads (thick filament) from binding with
complementary sites on the actin of the thin filament.
When Ca2+ is released into the cytosol (yellow circles),
it binds to troponin (light blue ovals) inducing a
conformation change in this protein. As troponin
changes shape, it alters the position of tropomyosin,
(purple ribbons) exposing the binding sites on the actin
molecules and allowing crossbridges to form.
sites on the actin molecule.
This blocks
crossbridges from forming, and hence no
contraction can take place. Only if troponin
repositions tropomyosin to expose the myosin
binding sites can crossbridge cycling occur.
This occurs in response to an action potential
being triggered in the skeletal muscle fiber,
which leads to a series of events collectively
called excitation-contraction coupling.
The contraction of a skeletal muscle fiber is
triggered by an action potential occurring in the
sarcolemma (plasma membrane) of that muscle
fiber (Fig 9.5). The action potential propagates
down the sarcolemma and is conducted down
transverse tubules into the interior of the cell.
This, in turn triggers the release of Ca2+ from the
sarcoplasmic
reticulum
(a
modified
endoplasmic reticulum) into the cytosol. The
Ca2+ binds to troponin on the thin filament and
causes it to undergo a conformational change
(Fig. 9.6). This change in the shape of troponin
shifts the position of tropomyosin on the thin
filaments, exposing binding sites for myosin on
the underlying actin and enabling crossbridge
formation (the bonding of myosin on the thick
filaments to actin on the thin filaments) to
commence.
Lab #9: Muscle Physiology
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Submaximal
Supramaximal
Stimulus Intensity
Muscle Tension
Subthreshold
Threshold
Stimulus
Maximal
Stimulus
Fig. 9.7. Whole muscle contraction in response
to stimuli of different strengths.
Muscle Twitch Parameters
A twitch is a muscle contraction that occurs in
response to a single, rapid stimulus that evokes a
single, isolated action potential in a muscle fiber.
Although single, isolated twitches are not in and
of themselves very useful for generating
controlled, coordinated movements needed for
maintaining homeostasis, observations of twitch
contractions present invaluable insights into the
basic physiology by which muscle fibers
generate tension.
Because the action potential is an “all or
none” response, the contraction of a muscle fiber
in response to a single action potential is
likewise an all or none response. Therefore,
there is a minimum stimulus strength that must
be applied to the muscle fiber in order to reach
threshold, evoke the action potential and, in turn,
induce the contraction.
Once the action
potential occurs, though, no further increase in
stimulus strength will increase the strength of
contraction, as the Ca2+ gates in the sarcoplasmic
reticulum are open for a fixed amount of time
once opened.
Individual muscle fibers respond to isolated
stimuli in an all or none fashion. However, a
muscle organ, such as the gastrocnemius muscle,
is composed of many individual muscle fibers.
By varying the number motor units (groups of
muscle fibers innervated by a singe somatic
motor neuron) contracting at a given time, the
amount of tension generated by the whole
muscle can vary. In one of the experiments we
are performing today, you will note that the
strength of the contraction varies with the
strength of the stimulus applied (Fig 9.7). This
does not violate the all or none principle.
Rather, as stimulus strength is being increased,
progressively more muscle fibers reach their
thresholds and contract. Thus, the change in
tension is due to the number of contracting
muscle fibers, not a change in how much tension
the individual fibers are generating. Note that
stimuli below the minimum strength needed to
trigger any of the muscle fibers to reach
threshold and undergo an action potential (i.e.,
subthreshold stimuli) will not trigger any
contraction in the muscle.
Threshold is
considered to be the level of stimulation required
to trigger the smallest measurable contraction
resulting from the excitation and contraction of
the first few muscle fibers. If stimulus is
increased above threshold into a range of
stimulus intensities called submaximal stimuli,
contraction strength will increase with stimulus
intensity as progressively more and more muscle
fibers in the muscle undergo contraction.
Finally, when stimulus strength is increased
above a certain level (maximal) no further
increase in tension occurs, as all muscle fibers in
the muscle are contracting.
A rather complex series of events occurs
within the time course of a single twitch. The
action potential is evoked upon application of
the stimulus. That action potential, in turn,
propagates down the length of the muscle fibers
and triggers the excitation-contraction coupling
process (release of Ca2+ from the sarcoplasmic
reticulum, binding of Ca2+ to troponin, etc.).
Once crossbridge cycling ensues, the muscle
fibers contract, generating tension. Tension
peaks, but then decreases as the activity of Ca2+
pumps in the sarcoplasmic reticulum reuptake
Ca2+from the cytosol, lowering the ability of
actin and myosin to form crossbridges, and
reducing tension generation as the fibers stretch
back to their original length. These three basic
stages (excitation-contraction coupling, tension
generation, and relaxation) correlate with three
different time phases during the twitch (Fig 9.8).
During the latent period (the time between the
Lab #9: Muscle Physiology
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Stimulus
Tension
Muscle Tension
Peak Tension
Stimulus
½ Peak Tension
½ Relaxation Time
Latent Period
Contraction Time
Baseline Tension
Tension
Stimulus
Total Relaxation Time
Tension
Fig. 9.8. Time intervals of a twitch contraction.
Stimulus
application of the stimulus and the onset of
contraction), excitation-contraction coupling
takes place. During the contraction time (the
time from the onset of contraction to peak
tension), crossbridge cycling occurs at a high
enough rate that the muscle fibers shorten.
During the relaxation time (from peak tension to
the point when tension returns to baseline), Ca2+
is being pumped back into the sarcoplasmic
reticulum, and the muscle is stretching back to
its original length. Since the duration of the
total relaxation time is often difficult to calculate
(since it is difficult to determine exactly when
tension returns to baseline levels), researchers
commonly use an alternate measurement, the ½
relaxation time, which is the duration it takes
for tension to drop from peak tension to ½ of
peak tension.
Summation and Tetanus
Observations of twitch contractions within
single muscle fibers or within whole muscle
organs can yield important insights into the basic
cellular processes involved in converting an
electrical signal into a mechanical response by
the muscle fibers.
However, with a few
exceptions, twitch-types of contractions are not
the typical type of contraction that skeletal
muscles inside the human body produce. That is
Fig. 9.9. Contractile response of muscle stimulated at
varying frequencies. Note the fusion of contractions
and the overall increase in tension generated with high
frequency stimulation.
because in order to enable coordinated body
movement and the maintenance of balance and
posture (the primary function of most skeletal
muscles), tension must be sustained beyond the
fraction of a second generated by a twitch.
Therefore, most skeletal muscle contractions in
the body are tetanic contractions.
The basis of “tetany”, or “tetanus” (not to be
confused with the disease commonly called
“lock jaw” caused by the bacterium Clostridium
tetani) within a skeletal muscle organ can be
somewhat confusing. Some authors believe that
tetany is due solely to the generation of twitches
by different groups of motor units occurring
asynchronously, so that as one group of motor
unit enters its relaxation phase, another is in its
contraction phase, etc., so that the sum of these
different units contracting is a smooth, steady
level of tension. This, however, presumes that
individual muscle fibers (or single motor units,
for that matter) are incapable of generating
sustained tension, which is incorrect—isolated
individual muscle fibers are able to generate
sustained levels of tension with high frequency
stimulation. To understand how, we need to
keep in mind that the electrical excitation of the
Lab #9: Muscle Physiology
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skeletal muscle (i.e., the action potential) and the
mechanical response of the muscle (tension
generation) do not have the same time courses.
It takes ~10 msec for an action potential to be
propagate down the length of a skeletal muscle
fiber in the frog gastrocnemius, whereas the total
time for a twitch contraction of the
gastrocnemius may be ~150 msec. Thus, many
action potentials can occur in the amount of time
needed for a single twitch. If a muscle that is
relaxing from a contraction is stimulated before
it fully relaxes, the sarcoplasmic reticulum will
release more Ca2+, and the cell will begin to
contract again without fully relaxing (See Fig
9.9). In effect, then, the twitches partially fused
together. If stimulated at progressively higher
frequency, the amount of relaxation that occurs
in between each “twitch” is progressively
reduced, until a steady state of tension (tetanus,
or tetany) is generated.
The tetanic contractions generated in today’s
experiment are caused by sustained, steady
levels of tension generated by individual muscle
fibers stimulated electrically at high frequency.
In most of the tetanic contractions in the body,
however, complete tetanus (contraction without
any relaxation) is not common. Most sustained
contractions are generated by a combination of
twitches and partial-tetanic contractions by
different motor units whose motor neurons are
stimulating the fibers at different intervals and at
different frequencies.
Interestingly, the amount of tension
generated during a tetanic contraction is often
substantially higher than that of a maximal
twitch. There are several reasons for this. First,
when a muscle begins to contract, some of the
tension generated by the muscle is absorbed by
stretching elastic elements within the muscle’s
attachments. This can reduce the total tension
generated on the attachments in a twitch
contraction whereas tetany, these elastic
elements are fully stretched and more tension is
exerted directly on the attachments. Secondly,
recall that each time the muscle fibers undergo
action potentials Ca2+ is released from the
sarcoplasmic reticulum.
The sarcoplasmic
reticulum begins to reabsorb this Ca2+almost as
soon as it is released, but it does take time to
fully recover all of the Ca2+. If the sarcoplasmic
reticulum is induced by another action potential
to release Ca2+ before it has fully recovered all
of the Ca2+previously released, then there will be
overall more Ca2+ in the cytosol during the
second contraction, more interaction between
actin and myosin, and a stronger resultant
contraction. Thus action potentials generated in
rapid succession can have a summation effect
on the strength of the contraction.
Functional Contraction Types
The contractions generated by skeletal muscles
are used for two basic functions: movement of
the body and maintaining position and
orientation of the body. Isotonic contractions
are those that result in the muscle shortening in
length, generating movement of a body part. In
order for an isotonic contraction to occur, the
muscle must contract with enough force to
overcome the load applied to the muscle.
Isometric contractions, in contrast, are
contractions where the muscle is contracting and
generating tension, but the muscle does not
shorten in length as the force generated by the
muscle is equal to the load place on the muscle.
Muscles that allow you maintain posture
generate isometric contractions to counteract the
force of gravity.
Electromyograms
The action potentials generated by contracting
muscle alter the electrical charge in the
surrounding extracellular fluid. These electrical
changes are conducted through body fluids, and
can be detected from the surface of the skin
using electrodes applied to the skin. A variety
of instruments can detect the differences in
charge between the electrodes, amplify them,
and generate recordings of these electrical
changes called electromyograms (EMGs).
EMGs are used diagnostically to detect damage
to muscle or to the neural pathways responsible
for triggering muscle contractions.
Lab #9: Muscle Physiology
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Experiment I. Measurements from Bullfrog Gastrocnemius Muscle Contractions.
In this experiment we will take direct measurements from a recording of actual frog muscle contractions
using the LabScribe software used to record from the iWorx physiography system.
A. Effects of Stimulus Strength on Tension Generation in Whole Muscle
1. Go to the computer screen, and be sure the software (LabScribe) is running. The top tracing will
display the voltage for the electric stimulus applied to the muscle (Fig 9.10). The lower tracing
displays the tension generated by the muscle (here expressed as voltage, but normally this would be
converted into some measurement of force). The display time should be set to 20 sec, meaning that
the 20 seconds of recording are displayed on the screen at any given time. If it is not, change it to 20
sec by selecting the EDIT menu from the top, then PREFERENCES, then enter the desired display
time in the middle box of the top line.
2. Scroll the recording to the right using the scroll bar at the bottom of the screen. Eventually you will
see a series of recordings where the stimuli and associated contractions from the muscle become
progressively stronger. These are recordings of twitches obtained by shocking the muscle with
different voltages. Each stimulus is preceded by a marker bar that crosses both recordings. At the
bottom of the screen are labels for each marker that provide the voltage of the electrical stimulus
applied to the muscle in each case (See Fig 9.10).
Stimulus voltages used to trigger the contractions
Fig 9.10. A series of recordings of twitch contractions recorded with the LabScribe software. The upper tracing records the
stimuli applied to the muscle (also marked at the bottom of the screen). The lower tracing records the twitch contractions.
Markers between recordings appear as brown vertical lines demarcated by the respective stimulus intensities at the bottom of
the screen. Measurement markers appear as blue vertical lines. Note that there is and “x” on the measurement marker where
it intersects the tracing for each recording. The difference in contraction tension (here recorded in volts, V2-V1) is located
just above the lower tracing to the right. The difference in time between markers (here recorded in hours:minutes:seconds,
T2-T1) is located at the top left corner of the screen just below the menus.
Lab #9: Muscle Physiology
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3. At the top of the screen is a row of clickable buttons. Click on the second to last button in that row
(the one that looks like this: ). You will now have two vertical blue bars crossing the screen. Note
that at the points where these blue lines cross the tracings for both the stimulus and muscle tension
they form a “×” icon. Also notice that as you change the position of the blue lines numerical values
in a couple of sets of readouts on your screen change as well: “T2-T1”, located at the top screen,
provides the time difference between the two points in your recording where you have positioned the
blue lines; “V2-V1”, located at the upper right corner of EACH of the two tracings, gives the
difference in voltage (amplitude) between the points demarcated with the blue lines.
4. Using the V2-V1 readout for the lower (Muscle) trace, we will measure the strength of muscle
contraction in response to different stimulus intensities. All measurements for this exercise will be
taken from the lower tracing. Using the pointer on the screen, left click and hold on one of the two
blue lines and drag it until it falls on the baseline area to the right of the recording somewhere
between the end of the twitch contraction and the marker for the next recording (See the example in
Fig. 9.10). Then left click on the other blue line and drag it until it falls at the peak of the twitch.
Record the strength of the contraction (voltage) from the V2-V1 readout for each of the twitches
(NOTE: record “0V” if there is no visible twitch recorded).
5. Consult your data sheet for additional questions.
B. Twitch Time Parameter Measurements
1. Reset the display time to 1 second. Under the EDIT menu at the top, select PREFERENCES, then
enter the display time (1 sec) in the middle box of the top line. Notice that this stretches out your
tracings so that the spike-like twitches seen earlier now appear as more curved waves (Fig 9.11).
Scroll through the tracing until you find a nice, robust twitch recording (e.g., at 1.5 or 2.0 V). Click
on the button at the top to bring up the two blue marker lines.
2. Measure the latent period of the twitch by placing one marker at the point on the top (stimulus)
recording right at the beginning of the square waveform and the other marker at the beginning of the
twitch on the lower tracing. Record the difference in time (T2-T1, top left corner). NOTE: The time
difference is given in hours:minutes:seconds. Record your measurements in milliseconds! WE
WILL DOCK POINTS FOR NOT EXPRESSING VALUES IN MILLISECONDS!!!
Fig 9.11. Position of measurement markers (in blue) for measurement of the latent period (left) and the contraction time
(right)
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Fig 9.12. Position of measurement markers (in blue) for measurement of the ½ relaxation time. First, position one marker at
the beginning of the contraction and the other at the peak of the contraction (left). Record the tension of the contraction (V2V1) calculate ½ of the contraction tension by dividing the peak tension by 2. Then, move the left hand marker to the right of
the peak until the tension reasing (V2-V1) is as close to the calculated value for ½ contraction tension as possible (right).
Then record the resultant time difference (T2-T1), which will be the ½ relaxation time.
3. Measure the contraction time by placing one marker at the beginning of the twitch and the other at the
highest point in the twitch.
Record the difference in time (T2-T1, top left corner).
4. To measure the ½ relaxation time (Fig 9.12), record the tension (V2-V1) of the contraction from the
onset of the contraction to the peak. Divide this number by 2 to calculate ½ peak tension. Then,
move the marker from the onset of the contraction and move it to the right of the peak until the V2V1 reading for the lower trace is as close to ½ peak tension as possible. Record the difference in time
(T2-T1, top left corner).
5. Consult your data sheet for additional questions.
C. Summation and Tetanus
1. Reset the display time to 10 seconds. Under the EDIT menu at the top, select PREFERENCES, then
enter the display time (10 sec) in the middle box of the top line. Scroll the recording to the right
using the scroll bar at the bottom of the screen until you reach the “1 Hz” marker. The markers from
here onwards indicate the frequency (Hz = #events/sec) that a 2V stimulus is being applied to the
muscle (Fig 9.13).
2. Place one of your two blue measurement markers in the baseline area to the right of the contraction
series between the contraction recording and the brown reference marker for the next recording (see
Fig 9.13 and note the position of the measurement markers with the recording for 10 Hz). Move the
other measurement marker across the peaks in the contraction and note the change in tension (V2-V1)
that occurs. Try to locate the highest point of tension in the recording (usually near the end of the
contraction) and record this value.
3. Consult your data sheet for additional questions.
Lab #9: Muscle Physiology
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Peak tension marker
Baseline marker
Fig 9.13. Recordings of muscle contractions evoked by series of stimuli at different frequencies.
Experiment II: Electromyogram
1. Place three adhesive disk electrodes in a row down the center of the inside of your lower arm.
2. Attach the EMG electrode cables to the
electrode disks in the following order (the
disks snap on to the cables):
1. Red (+)– most proximal (closest
to your elbow)
2. White (-) – middle
3. Black (gnd) – most distal (closest
to your wrist)
3. Hold the dynamometer in the hand of
the arm to which you have attached the
electrodes.
4. Click START in the upper right corner
of the screen.
Fig 9.14. EMG tracing illustrating motor unit recruitment. Note that
increasing the number of active motor units (indicated in the EMG
recording at the top) leads to an increase in tension generation (from
the dynamometer recording below).
5. Squeeze lightly on the dynamometer,
then release. Note that a light distortion
occurs in the upper tracing (see Fig 9.14).
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This is the electromyogram—a recording of the electrical activity of the muscle. Also note the
upward deflection of the lower tracing, which is a recording of the pressure being applied to the
dynamometer.
6. Squeeze the dynamometer again, slightly harder than before. Notice that the increased strength of
contraction is accompanied by an increase in the amount of distortion in the EMG tracing. This is
because of motor unit recruitment—you are activating more motor units to increase the overall
strength of contraction in the muscle organs, thus creating a larger electrical change in the body
fluids. Repeat this procedure several times, increasing how strongly you squeeze each time (Fig
9.14).
7. Put down the dynamometer. Extend the fingers into a relaxed position. Flex each finger individually
for one second (generating an isotonic contraction) then extend the finger back to its original
position. Does the flexion of some fingers produce an EMG signal whereas flexion of others does
not?
8. Produce an isometric contraction by placing your extended fingers against the underside of the
countertop. Flex each finger against the countertop. Note that when the muscles contract an EMG
signal is produced, even if the muscle itself does not shorten.
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