1

SimMuscle
Physiological experiments on isolated frog muscle
in a virtual laboratory

Table of Contents
Aims ........................................................................................................ 1
Required background knowledge ................................................................. 2
Preliminary excercises................................................................................ 3
1. The Virtual Laboratory........................................................................ 4
The suspension device and mechano-electrical converter ............................... 5
Stimulus apparatus.................................................................................... 5
Oscillograph.............................................................................................. 6
2. The Experiments ................................................................................ 7
Stimulus-Dependency of the Muscle Twitch .................................................. 7
Superposition of Double Stimuli .................................................................. 8
Tetanic Contraction ................................................................................... 8
Passive stretch-force curve of skeletal muscle ............................................... 9
Muscle fatigue ........................................................................................... 9
3. Practical notes on experimentation –in real and virtual lab.............. 11
Measurement of the nerve-muscle preparation............................................ 11
Muscle pre-stretching .............................................................................. 11
Muscle fatigue ......................................................................................... 12
Muscle deformation ................................................................................. 12
Supra-maximal stimuli of muscle ............................................................... 12
Single twitches versus tetanic contractions ................................................. 13
Isometric and isotonic contractions ............................................................ 13
The physiological diversity of the preparation.............................................. 13
4. Physiological basis ........................................................................... 14
The actin-myosin cross-bridging cycle ........................................................ 14
Action potentials and Ca2+ - controlling muscle contractions ......................... 15
Superposition of single twitches and tetanic contractions. ............................. 16
Effect of Pre-stretching Muscle .................................................................. 17
Muscle fatigue ......................................................................................... 18
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Aims
Most of the recordings that you will make in this practical lesson will
generate diagrams that you will also find in physiology textbooks. You
will find similar diagrams in recordings of cardiac muscle contractions.
These diagrams, such as isometric and isotonic maxima, are usually not
easy to understand. However, once you have produced these diagrams
through your own experiments, you should no longer have much
difficulty understanding what they represent.

By proper physiological experimentation and appropriate

documentation of your results, you should learn how macroscopic
observations reflect underlying physiological and anatomical conditions;
this is a principle that routinely applies in everyday clinical practice.

All the background knowledge that you require for performing this
practical lesson is described in detail in textbooks while, here, you just
need to make the right associations between theoretical facts and
experimental observations.

Required background knowledge

Morphological/functional organization: muscle fibers, myofibrils,
sarcomere, filaments.

Electromechanical coupling: action potentials, ryanodine and

2+
dihydropyridine receptors, the role of the calcium ion (Ca ), the actin-
myosin cross-linking cycle, the role of ATP.

Innervation: motor end-plate, motor units, muscle fiber recruitment.

Mechanics: forms of muscle contraction, myoelectric superposition and

tetanus, effect of muscle pre-stretching, isometric and isotonic maxima,
muscle fatigue.
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Preliminary excercises

Q: 1. Explain the following terms, using examples from daily life:

isotonic, isometric and auxotonic contractions; support and
stop twitching of muscle.
Q: 2. Describe the anatomical and functional structure of the
contractile apparatus of the skeletal muscle.
Q: 3. Describe the underlying molecular mechanism of muscle
contraction.
Q: 4. Why does the muscle power change with different degrees of
pre-stretching?
Q: 5. What is the relationship between the isometric curve (muscle
force at maximal tetanic contraction vs. relative muscle
length) and sarcomere length?
Q: 6. Describe the processes of synaptic transmission at the
muscle end-plate, from the nerve action potential to that of
the muscle.
Q: 7. Describe electromechanical coupling, from muscle action
potential to muscle contraction.
Q: 8. Draw the approximate time-dependent relationship between
muscle action potential, the resultant (single) muscle
contraction and the intracellular Ca2+ levels.
Q: 9. Name the neurotransmitter and its target receptor in the
muscle end-plate, as well as the substances that can be used
to block the neuromuscular transmission.
Q: 10. Name the two factors that physiologically control the muscle
force. (In the experimental procedures, which of the
stimulant parameters correspond to these factors?)
Q: 11. What effect does a fall in energy substrate (ATP, creatine
phosphate) have on the twitch process?
Q: 12. Explain the cellular processes that underlie a physiological
tetanus of skeletal muscle and explain why this does not
occur in cardiac muscle.
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1. The Virtual Laboratory

The virtual “SimMuscle” laboratory contains all the apparatus, in a

simplified but quite realistic form, that you will need to perform the
experiments. On one side, there is the 2-channel memory oscilloscope
for displaying the stimulus and the resultant muscle contraction. On the
other side, there is stimulus apparatus. In the middle, you will find
the suspension device from which the nerve-muscle preparation will
be suspended. This device includes an integrated mechano-electrical
converter for generating electric potential from muscle power and
muscle length changes.

Two nerve-muscle preparations will be made available to you. Although

this virtual laboratory allows you to avoid preparing the isolated tissue,
you may watch a video of one such preparation being performed; you
can access this video via the Info bar below the laboratory display. Also
via this Info bar, you can access a copy of this handbook (“Tutorial”)
and the experimental report form (“Protocol”). The report form allows
you to record your measurements and the experimental settings of the
stimulus device, and to plot the response curves. Alternatively, you
may enter your data in to the excel form that is integrated in to the
virtual laboratory and, in this way, obtain automatically-derived
response curves.
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The suspension device and mechano-electrical converter

The preparation needs to placed on the suspension apparatus; you do
this by clicking on the preparation, which is lying in nutrient medium in
the petri dish, and, with the mouse, drag the preparation to the
suspension apparatus. The preparation will be fixed in place via a piece
of bone above and via a small moveable loop below. The nerve of the
preparation lies over the two stimulus electrodes that are connected via
a double-cabled wire to the stimulus machine.

The position of the moveable loop indicates any change in muscle

length and this will be shown on the transmitter, such as the change in
muscle length after hanging a weight from the muscle. To hang a
weight from the muscle, click on the weight and drag it to the
preparation, placing the hook of the weight through the loop. You can
then hook on more weights, as desired. With increasing weight load,
the muscle will stretch but by a reduced degree with each similar
increase in weight (see exercise 4).

The integrated transmitter of the suspension device converts the

mechanical parameters of force and length change in to electrical
potentials that are transmitted to the oscilloscope, via the double-
cabled wire (blue). The conversion factors are fixed at 50 mV per N
(force) and 50 mV per mm (length change).

Using the toggle switch on the transmitter, (“Lock” or “Free”), you can
set, by mouse klick, whether force or muscle length are to be measured
(setting is indicated also by the green light): with “Lock”, the position
of the loop is fixed and there is no macroscopic change in muscle length
with a stimulus, which would be cause a isometric contraction; with
“Free”, the muscle can move freely and, after a stimulus, an isotonic
contraction occurs.

When the switch “ZERO ADJ” is activated (displayed in blue text), the
actual muscle force or length will be registered as the respective “zero”
value. If this switch is not active, then with every addition or removal of
weight, a new baseline value will be registered, with a corresponding
shift in the baseline displayed on oscilloscope. This setting can be used
to derive the passive stretch relationship of the muscle.

Stimulus apparatus
The stimulus apparatus delivers voltage pulses of fixed duration (1 ms).
The strength of the pulse (“Amplitude”) can be set using the upper
slide-control, while the lower slide control (“Period”) allows the
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frequency of delivery of more than one pulse to the muscle, if

required, by setting the time period between pulses. The “Mode”
switches allow selection of delivery of a single pulse (“Single”) or a
double pulse (“Double”) or a series of pulses (“Train”). For a series
of pulse, the number of pulses needs to be selected (“Counts”). When
all settings are selected, the stimulus is delivery (“Stimulation”) by
pressing the red button (“Start”).

The stimulus is delivered to the preparation via a double-cabled wire

and the nerve is activated as the impulse reaches the electrodes. The
stimulus also delivers a parallel signal to the oscilloscope via the green
cable to channel 1 of the oscilloscope. An additional cable (yellow)
connects the “Trig.Out” of the stimulus apparatus to the “Ext.Sig” of
the oscilloscope and provides a signal to trigger the oscilloscope to
provide a recording only during stimulation of the preparation.

Oscillograph
In order to obtain good recordings on the oscilloscope, the appropriate
scale settings need to be made. This is done by setting the
amplification of the stimulus and muscle contraction signals using the
knobs, “Channel 1” (mV/division (div)) and “Channel 2” (mV/div),
respectively. The time interval is the same for both channels and is set
by the knob “Time Base” (ms/div), for example, to 20 ms/div, which
would provide 10 sections over the width of the oscilloscope screen
(=200 ms). The zero line for each channel is highlighted and can be
shifted using the mouse.

It is adviced to have channel 2 on the first line at the bottom of the

screen as the muscle force and length changes (shortening) will be
projected upwards on the screen, with the exception of the passive
stretch-force curve. The channel 1 carries the stimulus information and
presents on the upper scale the time intervals of the impulses and the
duration of a series of impulses.

Normally, as one set of recordings starts, the oscilloscope removes

from the display the previous recording. However, by activating the
“Store” switch, this is prevented and a series of recordings can be
displayed on the screen. By activating the “Clear” switch, the screen
presentation is removed and no longer displayed. The switch “Undo”
allows the last recording to be removed from the display, for example,
in order to correct the settings and perform a re-run.
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2. The Experiments
In principle these experiments may be performed in any order.
However, they are presented here in an order that allows specific
aspects to be demonstrated step-by-step. In a first series of
experiments, you examine the strength of single twitches as a function of
the stimulus-strength. Then you demonstrate the time-dependent
summation of muscle contractions with superposition of double stimuli
and the development of tetanic contraction on trains of stimuli. The
second series of experiments begins with recording the passive stretch-
force curve of a skeletal muscle from which then the curves of isometric
and isotonic maxima can be determined and plotted in a way as they
typically are illustrated in conventional text-books. Finally, you can
examine the effects of muscle fatigue

Stimulus-Dependency of the Muscle Twitch

As described earlier, the strength of a single skeletal muscle twitch is
largely determined by the number of muscle fibers that are involved,
which in turn depends on the number of neurones that have reached
activation threshold; in other words the strength of contraction depends
on the number of motor units that have been activated. With a fixed
duration of stimulus (1 ms), the strength of a single muscle twitch is
exclusively a function of stimulus intensity.

From experience with this experimental set-up, the most powerful

contractions that provide the most reproducible results can be obtained
when the muscle is pre-stretched with 50 or 100 gram (weights 1-2).
Your measurements should start with stimuli of low amplitude (about
50 mV), followed by increments of about 50 mV until the muscle
contraction becomes visible in the display of the oscilloscope. You
should then reduce the stimulus step-wise in order to accurately detect
the minimum threshold (first visible contraction). The upper threshold
is reached when there is no further increase in contraction with
increased stimulus intensity. You should notice it is quite difficult to
accurately detect the lower and upper thresholds of the stimulus-
dependent contractions of your preparation. You should than make
recordings at suitable intermediate stimulus values in order to obtain
data points that span the lower and upper thresholds. You can than plot
the values and obtain the stimulus intensity vs. contraction strength
curve for your preparation. The plot should include values that are sub-
threshold (stimuli that failed to evoke visible contractions) and values
above the saturation or upper threshold (stimuli that failed to evoke
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any visible increase in contraction strength). You should also include

representative plots of single contractions in your experimental log.

Superposition of Double Stimuli

When successive stimuli are evoked within a short time interval, the 2
individual contractions will overlap (Superposition) if the second
stimulus is evoked before the previous contraction has faded. This you
can demonstrate by selecting a double stimulus (set mode switch to
“Twin”) and observe the contractions evoked after setting different
values for the time interval (PERIOD) between the 2 stimuli. You should
start your recordings with the time interval greater than the duration of
a single muscle twitch, which is 200 to 300 ms, and then reduce the
interval step-wise. An attempt should be made to determine the
stimulus interval at which a maximal superposition is observed.
Recordings of isolated single twitches and at about half-maximal
superposition should be entered in your experimental log. In the STORE
mode, you can select the appropriate time and amplitude settings.

Tetanic Contraction
The physiologically relevant form of skeletal muscle contraction is the
tetanic contraction. In vivo, muscle fibers of the motor neurons are not
activated by single action potentials but by prolonged bursts of action
potentials that evoke superimposed single twitch contractions (see 0
Superposition) to form a more-or-less single smooth contraction of the
skeletal muscle. In the following, you should aim to generate recordings
that range from a single contraction, through transitional stages of
superposition, to a smooth tetanus. The stimulus pulse should be fixed
at a level that evokes the same supra-maximal contraction but varied
in regard to impulse frequency. You can use either isotonic or isometric
conditions. For you documentation, you should have recordings of an
incomplete and a complete tetanic contraction (likely to occur at 10 Hz
and 20 Hz), and of a sequence of still well separated single contractions
(likely to occur at 4 Hz or 5 Hz), preferably with all three recordings on
the same oscilloscope display (using the Store mode). You must note
that the gain setting should allow for the tetanic stimuli
causing about three-times greater muscle force or change in the length
than that of a single muscle twitch. A suitable time scale setting is 200
ms/div, which gives a full oscilloscope display setting of 2 s. The
number of pulse stimuli should be selected so that the entire sequence
is displayed. For 2 s and 5 Hz, this would be 10 pulses. In order that
the muscle relaxation is still clearly visible, the total duration of
stimulus should be shortened by about 20% (which, in this example,
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reducing by 2 pulses to a toal of 8). For each doubling of the frequency,

you can double the number of pulses, such as by applying 16 and 32
pulses at 10 and 20 Hz, respectively.

Passive stretch-force curve of skeletal muscle

Because it is elastic, a muscle will change in length as it is stretched.
But unlike an ideal spring, the changes in length are not proportional to
the force of the stretch. You can demonstrate this by adding an
increasing number of weights to the non-stimulated (passive) muscle
preparation and record the change in length.

You should note that this can actually be done directly using the
oscilloscope. The stimulus intensity should be to 0 mV and channel 2
should be set at maximum. Without activating a zero adjustment
(ZERO ADJ), each additional weight will be observed to cause a shift in
the baseline of channel 2 and this shift directly relates to the change in
muscle length and can be used to generate the stretch-force curve.

Enter in Table 4, the weight force Fp [N] and the intrinsic passive
length change ΔLp [mm]. Then measure for each degree of pre-
stretching, the active forces ΔFa [N] and length changes ΔLa [mm] at
each single muscle twitch and enter the values in the table.

You can begin to create diagram 4, simply by using the force and
length changes of the active contractions (upper diagrams). The zero
point that is indicated on the abscissa marks the initial length. It is not
identical with the zero point of your coordinate system because you
have to consider muscle shortening, ie negative values of ΔL.

In the 4 lower diagrams, the passive stretch-force curves are plotted

and are, of course, identical. In order to create the classic isometric
curves and isotonic maxima, you need to add to these passive values,
the values of active force development (isometric maxima) or subtract
the values of active muscle shortening (isotonic maxima). The resulting
values of the isometric and isotonic maxima can be entered in the
appropriate columns of the table. You should add labelling to the
diagrams so as to achieve clarity (look for examples in your physiology
textbooks), such as indicating the different curves.

Muscle fatigue
The aim of this experiment is to directly compare the contraction curves
of a fresh and a fatigued muscle. The contrast is most marked in the
freely-suspended muscle (isotonic contractions). Again, you should pre-
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stretch the muscle (with one or two weights) as this optimally reflects
the normal physiological condition of skeletal muscle. Then, by adding
more weights to the muscle, you can quickly cause muscle fatigue.

Muscle fatigue can be demonstrated in different ways. You could

compare single contractions before and after fatigue is induced by a
prolonged tetanic stimulation. The oscilloscope should be set at 50
mV/div and 50 ms/div so that longer single muscle twitches can be
displayed. Also, you can detect on the oscilloscope muscle fatigue
developing during a tetanic contraction; but you would have to
temporarily reduce the sensitivity (200 mV/div) and slow the time
scale. On applying 200 pulses at a frequency of 50 Hz (total time 200 x
20 ms = 4000 ms), 500 ms/div (display time span of 5000 ms) is
required for the contraction to be fully observed on the display.

During a tetanic stimulation, the declining force of contraction can be

observed through progressive changes in the single twitches, in
particular, the significantly reduced rate of contraction and relaxation.
These changes, including decreasing contraction amplitude, can also be
illustrated by simply making a continuous recording of a prolonged
series of pulse stimuli. This should be over an extended period of time,
starting separate single twitches, with a setting of 200 ms. As muscle
fatigue develops after about 50 pulses, these individual twitches
become flatter and wider and begin to overlap. With a time setting of
50 ms/div 2 muscle twitch curves can be selected and superimposed
and so demonstrate this effect of muscle fatigue.
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3. Practical notes on experimentation

– in the virtual and real laboratory

Measurement of the nerve-muscle preparation

For recording skeletal muscle contractions, nerve-muscle preparations
are preferred for practical reasons, particularly relating to the need to
evoke muscle contractions electrically. This is normally achieved by
stimulating the nerve that supplies the muscle. In principle, the muscle
could be directly stimulated electrically by, for example, placing
electrodes in the form of a loop around the muscle. However, this
would require such a large stimulus intensity, to ensure activation of
the innermost muscle fibers, that the muscle fibers closest to the
electrode would be damaged. Also, because the position of the
electrodes will move with each muscle movement, the reproducibility of
the response is not guaranteed.

In preparing the nerve-muscle preparation you should make sure that a

stump of the femur remains, as this allows the preparation to be fixed
to the suspension device by attaching it via this piece of bone and
avoiding damaging the muscle or nerve. Particularly important is that
the nerve is comparatively thin and, therefore, allows all the neuronal
fibers (even the thick fibers of alpha-motor neurones) of the nerve to
be activated by a small and non-harmful current. The nerve can then
be loosely placed above the suspended preparation and in contact with
the stimulating electrodes, without interfering with the position of the
muscle and affecting the contraction. Thus, indirect electrical
stimulation allows the target tissue (the muscle) to be physically
unhindered during activation. It should be noted that, in contrast to
other physiological experiments involving nerve and muscle, here
mechanical parameters are being recorded, not electrical parameters,
and their response time-course is much longer.

Muscle pre-stretching
It is important to keep in mind that a special feature of skeletal muscle
in situ is that, in its passive state, it is pre-stretched. This means that,
when isolated from the body, skeletal muscle contracts and needs to
pre-stretched, by attaching weights, in order to exhibit physiologically-
relevant contractions of appropriate force. Tests can be performed on
isolated muscle to determine the range of optimal pre-stretching and to
generate the curves of isometric and isotonic maxima. Experience has
shown that in regard to the isolated gastrocnemius muscle of the
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clawed frog, pre-extension using a weight of 50 or 100 g provides the

physiological length and allows the muscle to exhibit its normal range
of contractions.

Muscle fatigue
Because of the absence of a blood supply and the normal level of
nutrient supply, the isolated skeletal muscle fatigues much more rapidly
than in situ, and once fatigued does not fully recover. For reproducible
and physiologically meaningful results, experiments on isolate muscle
must be performed systematically and unnecessary repetitive
contractions and prolonged tetanic contractions should be avoided.
Although there is short-term fatigue, from which the muscle may
recover in a few minutes, there is usually some residual fatigue that
remains. Returning the preparation, temporarily, to the Petri dish
allows the preparation to recover to some degree. In the virtual
laboratory, returning the preparation to the petri dish leads to an
immediate and complete recovery and so guarantees the comparability
of the experimental results.

Muscle deformation
Skeletal muscle is not an ideal elastic mechanical structure and also has
plastic components. This can lead to the muscle failing to return to its
original starting length after excessive and prolonged contractions. Also
this deformation affects the reproducibility of the experimental results.
In this virtual laboratory, these plastic effects were not taken into
account in the computer simulation. This is because muscle
deformation is not a physiological phenomenon but an artifact of the
isolated stated of the real experimental model. For this reason, in the
real isolated frog muscle preparation, it needs to be ensured that you
do not strain the muscles for too long and with too many weights, such
as in experiments aiming to derive the curves of isometric and isotonic
maxima.

Supra-maximal stimuli of muscle

Besides in experiment 1, you should always apply supra-maximal
stimuli, which means stimuli of strengths that are well above the
maximum threshold, which you determined in the first experiment. This
is important under real conditions to ensure that the stimulus always
evokes a similar response because small changes, such as drying of the
nerves, will occur that will slightly affect the stimulus conditions and
more so at sub-maximal stimuli.
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Single twitches versus tetanic contractions

In physiology textbooks, curves of isometric and isotonic maxima are
usually shown for measurements of tetanic contractions in individual
muscle cells. For a student experiment, the preparation of individual
muscle cells is too difficult and time-consuming. In experiments on
single muscle cells, fatigue is reached much more slowly than in
isolated whole muscle preparations. This is because of the easier access
of nutrients which encounter much shorter diffusion paths in the
isolated cell than in isolated whole muscle preparations. Thus isolated
cells are amenable to using tetanic contractions. In contrast, in isolated
whole muscle preparations, it is preferred to perform single twitches
rather than tetanic contractions in order to avoid excessive fatigue.

Isometric and isotonic contractions

In most of the experiments, it is irrelevant whether isometric or isotonic
contractions are performed; the one exception is experiment 0 0 which
aims to demonstrate the generation of isometric and isotonic maxima.
However, for demonstrative reasons, isotonic measurements have the
advantage that the muscle contractions are clearly evident in the
muscle, itself.

The physiological diversity of the preparation

As applies to all biological preparations, there is variation between
different preparations of the gastrocnemius muscle of the clawed frog,
in regard to their response behaviour. This is due to biological variation
arising from differences in anatomical and physiological features, such
as muscle size. Additionally, there is experimental variation that affects
measured response values, which can be crucially affected by, for
example, the positioning of the specimen in the measuring apparatus,
of the sensor and of the stimulus electrodes. A further source of
variability in isolated preparations is the quality of the preparation. For
example, if a lot of connective tissue is left around the nerve, greater
stimulation currents will be required than with a well-exposed nerve.
However, too much effort to expose the nerve may lead to nerve
damage and reduce the efficient of some of the motor units and cause
smaller than usual maximum contractions.
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4. Physiological basis

In order to perform the practical class

successfully and productively, you require a
sound knowledge of the basic physiology of
skeletal muscle, which you should have
acquired from your physiology textbook
and/or lectures. In the following, specific
aspects of muscle physiology are presented
that should be well-known to you but
perhaps not fully understood in regard to
their particular relevance to the process of
muscle contraction.

The actin-myosin cross-bridging cycle

The contraction of a muscle cell depends not
on the shortening of individual molecules but
the sliding of molecules over one another.
This is the general principle of contraction in
all types of muscle and involves the filament
molecules, actin and myosin, sliding over
each other. This shortens the length of the
sarcomere, which is the anatomical sub-unit of
the muscle cell that lies between the Z-
discs. The sliding of the actin and myosin
filaments is initiated by a Ca2+-dependent
process. Calcium binds to a site on the Figure 1: The Actin-Myosin Cross-
Bridge-Cycle, illustrated by the
actin filament, causing a conformational movement of a single actin
chain in the actin molecule that exposes a filament. You will find an
site for binding of the adjacent myosin animation at www.virtual-
physiology.de.
filament. As myosin binds to actin, a
molecule of ATP splits to ADP and inorganic phosphate and provides the
energy that is used to turn the neck of the myosin. This causes a shifting of
the myosin filament along the actin filament (ratchet-like) before another ATP
molecule becomes bound to myosin, causing a further conformational change
in myosin and releasing it from actin. In the continued presence of Ca2+
myosin binds again to actin and this cross-bridging cycling continues. This
process is shown in figure 1 for a single myosin filament; the more myosin
filaments involved the more observable macroscopically is the development of
the muscle contraction.
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Action potentials and Ca2+ - controlling muscle contractions

Skeletal muscle contrac-

tion is evoked by an action
potential across the
muscle cell membrane,
which is in turn evoked in
the motor end-plate in
response to an action
potential in an alpha-
motor neuron arriving at
the motor end-plate.

The motor end-plate is a

specialized synaptic
structure that, unlike other Figure 2: Control of skeletal muscle contraction.
On arrival of a muscle action potential in the transverse
synapses, allows one post-
tubules (T-tubules), voltage-dependent Ca2+ channels
synaptic action potential to (dihydropyridine receptors, localized at intervals in
be evoked in response to membrane of the T-tubules) are activated and, in turn,
each pre-synaptic action adjacent Ca2+-dependent Ca2+-channels (ryanodine
potential that arrives receptors) in the terminal cisternae of the longitudinal
system (sarcoplasmic reticulum) are opened. With the
there. In the experiments influx of Ca2+ and its accumulation in the cytoplasm,
to be performed here, it is Ca2+ binds to troponin C of the actin binding sites for
possible to observe that a myosin heads and so starts the cross-bridge cycle.
very brief stimulus, one Simultaneously, Ca2+ is pumped by Ca2+-ATPases back
into sarcoplasmic reticulum.
that evokes for sure not
more than a single pre-synaptic
action potential, is sufficient to
cause a single
muscle twitch.

The time-span of a muscle

contraction is significantly longer
than the time-span of the action
potential. For example, an action
potential of 1-2 milliseconds can
cause a muscle twitch lasting 100-200 milliseconds. This is because the actin-
myosin cross-bridging cycling is Figure 3: The different time scales of a muscle
not coupled directly to the muscle action potential, intracellular Ca2+ and increase
membrane action potential but of muscle contraction by a single muscle twitch.
The muscle contraction takes significantly
indirectly via the action potential- longer than the action potential that triggers it,
induced increase in the mainly due to the time lag in intracellular Ca2+
accumulation and the reverse pumping of Ca2+.
intracellular Ca2+ concentrations. Muscle contraction is also slowed by the
processes of the cross-bridges cycle and the
mechanical properties of the muscle fibers
(their elasticity and the damping effect of the
surrounding fluid).
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The action potential of the muscle fiber membrane is carried along the length
of the muscle fiber by the membrane of the T-tubules that penetrate the fiber
as invaginations of the muscle cell membrane (sarcolemma). The sarcolemma
comes in to close proximity to the membrane of the intracellular structure,
the sarcoplasmic reticulum, which stores high amounts of Ca2+ ions. By
activating specific proteins (dihydropyridine receptors/ryanodine receptors)
that are positioned at intervals to form molecular bridges between the
sarcolemma and sarcoplasmic reticulum, an action potential (in the
sarcolemma) causes extracellular Ca2+ to enter the muscle cell and
intracellular, stored Ca2+ to be released (from the sarcoplasmic reticulum)
into the cytoplasm of the muscle. High Ca2+ concentrations (10-7 moles/L) are
required in the muscle cytoplasm to activate cross-bridge cycling. As
cytoplasmic Ca2+ concentrations increase, the processes that pump Ca2+ back
in to the sarcoplasmic reticulum are activated causing the cytoplasmic Ca2+
concentration to decrease. The processes controlling these fluxes of Ca2+
across the sarcoplasmic reticulum and that produce a “wave” of increased
cytoplasmic Ca2+ require much more time than the duration of an action
potential. In turn, the mechanical processes of actin-myosin sliding and
muscle contraction and relaxation require much more time than the processes
of Ca2+ release and re-absorption across the sarcoplasmic reticulum. A
significant component of time required for a muscle contraction and
relaxation in response to a single action potential is the delay caused by the
combined effect of the elasticity of the muscle filaments and the mechanical
resistance provided by the surrounding fluid. This can be seen as analogous
to the response of a shock-absorber on a car; in response to a brief but
powerful pressure applied to a car wing, the shock-absorber causes it to
return to its original position without an over-swing but with a detectable
delay.

It is important to realize here that the muscle forces or changes in muscle

length that you can observe and measure are different to what is shown in a
myogram. A myogram measures actual muscle action potentials, which are
electric parameters, and not force or changes in length, which are mechanical
parameters.

Superposition of single twitches and tetanic contractions.

The difference in duration of the short action potential and the much longer
muscle twitch underlies the development of a tetanic contraction from single
twitches. Action potentials can be generated at intervals that are much
shorter than the duration of a single muscle twitch. If a second action
 17

potential arrives before the previously evoked muscle twitch has subsided, a
second muscle contraction will be added on top of the first contraction. When
several action potentials arrive in a short period of time, a tetanic contraction
will be formed by the single contractions superimposing and without a single
contraction being detectable.

The muscle force resulting from repeated action potentials is not proportional
to the frequency of action potentials for a number of reasons: (i) the elastic
resistance of muscle fibers increases, (ii) an equilibrium will be reached
between Ca2+ release and Ca2+ re-absorption, (iii) all the Ca2+ binding-sites
will eventually become saturated, and (iv) the rate of cross-bridge cycling will
reach a maximum, above which no further increase is possible. The force of a
tetanic contraction is usually never more than 3-fold greater than that of a
single muscle twitch. Much more important for increases in the force of the
whole muscle is the recruitment of motor units. Under physiological
conditions, a relatively low frequency of action potentials can lead to a tetanic
muscle contraction through overlapping contraction of several motor units
with alternating innervations. This is not observed in the isolated muscle
preparation because the action potential, in this case, is evoked by an
external stimulus.

Figure 4: single muscle twitch; incomplete superposition of 2 and 6 muscle twitches; and
complete (smooth) tetanus.

It should be noted here why tetanus cannot occur in heart muscle. This can
be easily explained by reference to the duration of events underlying heart
muscle contractions. The heart muscle action potential lasts 300-400
milliseconds (ms) and is of a longer duration than the heart muscle
contraction. Therefore, after the cardiac muscle action potential is over, the
cardiac muscle contraction is already over. So, if a second action potential
was to arrive immediately, there would be no previous contraction present on
which a second contraction could superimpose. Thus it is not the refractory
period, as one often written in physiology text books, that prevents
superposition of heart contractions as occurs in the skeletal muscle.
Additionally, the action potential of the skeletal muscle has a refractory period
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due to the different time relations between duration of action potentials and
single twitches. (For the occurrence of sustained but uncoordinated heart
contractions that occur during cardiac arrest see “SimHeart”).

Effect of Pre-stretching Muscle

The contraction strength of a muscle is very dependent on the degree of pre-

stretching but how contraction strength is affected by pre-stretching varies
between different types of muscle. The effect of pre-stretching can be readily
observed in the isolated skeletal muscle. When a skeletal muscle is detached
from a tendon, it is clearly seen to become shorter. If the isolated skeletal
muscle is then stretched by attaching a weight, as will be seen in this
practical class, it can return to its in vivo length and it acquires a contraction
strength that is close to that which it had in vivo.

Figure 6: Force development in a skeletal muscle fiber in relation to sarcomere length

(above left). The maximum force is set to 100%. Under physiological conditions, muscle
contractions occur within a narrow range around the maximum force. Above right is
shown the optimal degree of actin-myosin interaction with moderate pre-stretching
(middle), compared to the overlap of actin filaments that occurs in the totally unstretched
muscle (top) and with severe over-extension with a proportion of myosin heads without
access to sites for actin binding (below). The lower diagrams show the corresponding
recordings of an isolated whole muscle (from the laboratory SimMuscle) with the different
degrees of pre-strain.
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This has a lot to do with the stretching of the skeletal muscle allowing a
maximal overlapping of the myosin and actin filaments, with optimal
stretching allowing each myosin head to have access to a binding site on
actin. Also, stretching the muscle increases the Ca2+ sensitivity of the myosin-
actin interaction. Without pre-stretching, the actin filaments overlap and
prevent myosin from binding or may even cause contractions in the wrong
direction. Over-stretching the muscle reduces the access of myosin heads to
actin.

It is important to note here that this phenomenon, as it is seen in vivo, differs

between skeletal muscle, heart muscle and smooth muscle (see also
SimHeart and SimVessel). Whereas the normal basal, unstimulated pre-
stretched state of skeletal muscle provides for maximal muscle strength,
heart muscle and smooth muscle have a considerable reserve of potential
muscle strength when in their basal state. The strength of the heart muscle is
increased as it is stretched during filling of a heart chamber (the Frank-
Starling mechanism) and intestinal and vascular smooth muscle only become
active as they are stretched in response to increased luminal pressure.

Muscle fatigue
The experience of muscle fatigue after prolonged strenuous exercise is a
common experience. Several factors are involved in the experience of muscle
fatigue. In real life, psychological factors mostly play a role in strenuous
exercise and allow the feeling of “fatigue” to set in before true muscle fatigue
occurs. Of course, with the isolated skeletal muscle such factors can be
ignored. However, muscle fatigue is reached much sooner in the isolated
skeletal muscle than in the skeletal muscle in situ. This is because of the
absence of a blood supply and consequent lack of sufficient energy supplies
and of adequate removal of metabolic waste products.

It is possible to recognize whether an isolated muscle is fresh or fatigued.

Figure 5: Single muscle twitch of an isolated skeletal muscle, before and after fatigue.
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Usually, muscle fatigue in an isolated skeletal muscle is reached after 100

stimulated tetanic contractions. The form of the muscle contraction is clearly
different when comparing recordings before and after 100 tetanic contractions
(Figure 3). Not only is there a reduction in muscle power but also a much
more prolonged contraction, which is usually observed before any clear
decline in muscle power is observable. Although much of what causes muscle
fatigue is unclear, the reduction in the intra-cellular levels of ATP is clearly an
important contributory factor. ATP is needed not only for cross-bridge cycling
(for releasing the myosin head from its binding site on actin and for inducing
the “bending” of the myosin head) but also for pumping Ca2+ back in to the
sarcoplasmic reticulum. A slowing down of both these processes would delay
muscle contraction and movement would be slow and imprecise. Those
myosin heads that are still bound to actin will not be able to contribute to
muscle contraction and muscle power would be lost. In the complete absence
of ATP, all the myosin heads will remain fixed to their binding sites on actin.
This is seen in the facial muscles of a corpse, causing the fixed stare. A deficit
of ATP in skeletal muscle may also cause muscle cramp.

There are several other candidate factors that may contribute to muscle
fatigue. These include the increased intracellular levels of ADP and inorganic
phosphate, which directly relates to the decreased ATP levels, and the
decrease in the pH of the muscle tissue. In particular, raised levels of
inorganic phosphate affect not only the cross-bridge cycling process but may
also slow the rate of Ca2+reabsorption. However, the underlying mechanisms
of these effects are unknown.