Download Turtle heart - Missouri State University

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Heart Muscle
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Background
The heart is composed of myocardial cells, which contract in
a coordinated fashion to pump blood around the body. The
pacemaker of the heart is the sinoatrial (SA) node, located
in the right atrium. The SA node contains modified muscle
cells that are autorhythmic (have a pacemaker potential).
An action potential from the SA node travels via gap
junctions to adjacent cells in the atria. The gap junctions,
which are part of the intercalated disks between adjacent
atrial cells, allow the action potential to move around both
atria like a wave, causing the atria to contract. The action
potential also spreads to the atrioventricular (AV) node,
which is also composed of specialized muscle fibers. The
action potential moves slowly along the electrical pathway in
the AV node, and then travels rapidly along the Bundle of
His and Purkinje Fibers to the fibers of the ventricle. The
slow transmission of the action potential through the AV
node insures that the ventricles contract after the atria. This
delay allows the ventricles to fill with blood from the atria
before the ventricles contract.
The contraction of the heart cells follows the sliding
filament model as described for skeletal muscle. Contraction
of the atrial cells force blood into the ventricle, and
contraction of the ventricle force blood out to the body.
Mammals have two ventricles, which pump blood to the
lungs and rest of body separately. The term “systole” refers
to the contraction phase of the heart, and “diastole” refers to
the relaxation phase of the heart cycle. Unless otherwise
indicated, these terms refer to the action of the ventricle.
Change number of channels (upper right hand corner)
to 4
Change Function to Stim 1
Change Name to Stimulator
6 Once you go back to the Main Screen, you will have to
minimize Channels 1 & 2
The Dissection
Hints
1 Keep the exposed heart moist with Ringer's solution until
the experiments have been completed.
2 Be prepared to do your experiments quickly and efficiently,
And then perform your data analysis after all of the exercises
have been completed.
Equipment Setup
1 Plug the DIN connector on the cable of the FT-100 force
transducer into Channel 3 of the iWorx unit (Figure 1).
2 Plug the BNC-double banana adapter into the positive
(red) and negative (black) sockets of the stimulator of the
iWorx unit. Check the side of the double banana adapter for
a tab. This is the side of adapter that goes into the negative
(black) socket of the stimulator.
3 Do not connect the stimulator cable to the adapter on the
stimulator of the iWorx unit until you reach Exercise #4
.
Figure 1: The equipment used to record heart mechanical contractions.
Pretend this is a picture of turtle.
Procedure
4 Arrange the clamps on the ring stand so that the string
from the heart will be at as low an angle as possible without
the pin/string hitting the sides of the hole. The blade of the
transducer should be perpendicular to the sting.
1 Place the recently-pithed turtle ventral surface up, in the
dissection tray. A hole saw was used to make a circular cut in
the plastron (ventral bony plate).
Start the Software
2 Lift up gently on the disk of bone, and use the scissors to cut
the connective tissue and remove this disk.
1 Click on the LabScribe icon on the Desktop.
2 Click on the Settings menu again and select the HeartMuscle settings file.
3 After a short time, LabScribe will appear on the computer
screen as configured by the HeartMuscle settings.
4 Close the pdf file that opened automatically.
5 We need to also display the stimulator output. To do this:
Click on Edit
From the pull down menu, chose Preferences
3 Locate the heart, which is covered by the pericardial sac.
Use forceps to grasp and lift up on the pericardial sac, not the
heart. Dissect away the pericardial sac from the heart, trying to
avoid the cutting of blood vessels in this sac. Do not cut any of
the vessels attached to the heart.
4 Use masking tape to secure the turtle to the tray, without
blocking the hole (Figure 2).
Force transducer
Choose a destination on the computer in which to save the
file(e.g. the iWorx or class folder). Click the Save button to
save the file (as an *.iwd file).
Exercise 2: Effects of Temperature
thread
Aim: To record changes in heart rate and force of contraction
after the heart is bathed in warm and cold Ringer’s solution.
Procedure
1 Click Start to begin recording. Click AutoScale to increase
the size of the deflection on the Main window.
Figure 2: Set up for turtle in dissecting tray ready for testing..
5 Use forceps to grasp the frenulum (a tendon at the apex of
the ventricle) and insert the heart pin thought the heart near
the frenulum, penetrating about 2 mm deep (Figure 3). The
cut the frenulum.
6 Tie the thread of the heart pin to the force transducer (the
thread should be as short as possible), and gently move the
ring stand so that the thread is taut and the heart is slightly
stretched.
2 Type “Room Temp Ringer’s” on the comment line to the
right of the Mark button. After recording about 15 seconds of
heart contractions, use a Pasteur pipet to apply about 20 drops
of frog Ringer's solution (at room temperature) to the heart.
Press the Enter key on the keyboard when the solution is
dropped on the heart, and record for about another 45 seconds
before clicking Stop.
3 Type “Cold Ringer's” on the comment line, and repeat this
60-second procedure with cold Ringers.
4 Repeat the procedure with warm Ringers.
5 Select Save in the File menu.
6 Flush the heart with room temperature Ringer’s solution.
A
A
Exercise 3: Effects of Drugs
Vent.
Aim: To monitor the effects of natural and pharmaceutical
chemicals on the amplitude and rate of heart contraction.
Procedure
1 Click Start to begin recording. Click AutoScale to increase
the size of the deflection on the Main window.
frenulum
Figure 3: The turtle heart with pin in place
Exercise 1: The Heart Rate and Force
Aim: To record the mechanical trace produced by the
contraction of a resting heart, and to determine the
resting heart rate.
Procedure
1 Click Start to begin recording. Click AutoScale to increase
the size of the deflection on the Main window.
2 Type “Resting” on the comment line to the right of the
Mark button. Press the Enter key on the keyboard to attach
the comment to the record.
2 Type “Epinephrine” on the comment line to the right of the
Mark button.
3 Record about 15 seconds of heart contractions, and then use
a Pasteur pipet to apply 10 drops of room temperature
epinephrine solution to the heart. Press the Enter key on
the keyboard when the solution is dropped on the heart.
Record the effects of epinephrine for anther 45 seconds. Then,
flush the heart with fresh, room temperature Ringer’s
solution until the heart rate returns to its resting value.
4 Click Stop to halt recording. Select Save in the File menu.
5 Repeat this 60-second procedure for acetylcholine.
Note: If the heart “arrests,” rinse the Acetylcholine solution off
the heart with fresh, room temperature Ringer’s solution. If the
heart is still “arrested” after 10 seconds, add two drops of
Epinephrine solution to the heart.
3 Record about 15 seconds of heart contractions. Then click
Stop to halt recording. Remember to moisten the heart with
frog Ringer’s solution at all times.
6 Repeat this 60-second procedure with atropine, but do not
rinse the heart with Ringers when this is over. Immediately
repeat the acetylcholine 60-second procedure. Then rinse with
room temperature Ringer’s solution until the heart rate returns
to its resting value.
4 Select Save As in the File menu, type a name for the file.
7 Finally repeat this 60-second procedure for nicotine.
Exercise 4: The Refractory Period of the Heart
Aim: To stimulate the ventricle to produce extra ventricular
contractions (extra-systoles), and to determine when the
heart is in its refractory period and unable to create extrasystoles.
Procedure
1 Adjust the bipolar stimulating electrodes on the ring stand
so the tips are touching either side of the ventricle, and the
ventricle is able to move up and down as it contracts, or hold
it steady by hand so that both prongs are touching the
ventricle.
2 Attach the BNC connector of the stimulator cable to the
adapter on the stimulator of the iWorx unit.
3 The stimulator has already been turned on and
programmed by the HeartMuscle setting file. The values of
the following parameters should be seen in the stimulator
control panel under the LabScribe toolbar:
• the stimulus amplitude is 4V.
• the stimulus delay is 50ms.
• the stimulus duration is 10ms.
• the frequency is 1.0Hz.
• the number of pulses is 30.
4 Click Start, and then AutoScale. Type “Refractory” on the
comment line. Press the Enter key on the keyboard. You
should see a rhythmic deflection on the trace.
5 Click Stop after recording for the 30 seconds that the heart
was stimulated. Examine your recording. Look for extra
ventricular beats (Figure 3). If you don’t see an
extrasystole, do the following, and repeat steps 4 and 5:
• Decrease the stimulus frequency.
6 Select Save in the File menu.
2 Click Start, and then AutoScale. Type “Tetanus” on the
comment line. After 10-15 seconds of recording, press the
Enter key on the keyboard. After the pulses stop, continue
recording for another 10-15 seconds.
Exercise 6: Effects of Stretching the Heart
Aim: To determine the relationship between length of cardiac
muscle and the force it produces (Starling’s Law of the heart).
Procedure
1 Remove the stimulating electrode for now. Make sure that
the thread from the heart to the force transducer is taunt and
the heart is only slightly stretched.
2 Start the recording and after 15 seconds, slide the turtle (and
tray) about 2 mm away from the force transducer, stretching
the heart. Record at this heart muscle length for about 15
second.
3 Slide the tray another 2mm, and repeat this every 15
seconds until you have slid it at least 5 times. The heart is
quite flexible, so this should not tear it.
4 After you complete the stretching, slide the tray back to
reduce the stretch on the heart
Exercise 7: Effects of a Ligature on the Heart
Aim: To observe the effects of blocking action potentials from
passing from the atria to the ventricle.
Procedure
1 Take a piece of thread about 12 inches long. Place the
center of the thread around the AV groove that separates
the ventricle from the atria.
2 Tie a single overhand knot in the thread, to form a loop
around the AV groove. Don’t tighten the loop at this time!
3 On the left and right sides of the heart, insert other thread
pieces through the ligature loop (Figure 4). On each side, you
will later bring the two ends together, and pull outward to undo
the ligature.
A
A
Vent.
Ligature release threads
Figure 3: Stimulation of the ventricle produced an extra contraction of the
ventricle when delivered at the appropriate time during the cardiac cycle.
Exercise 5: Effects of a Tetanizing Stimulation
Aim: To determine whether tetanus can be induced in
cardiac muscle, as seen in skeletal muscle.
Procedure
1 Adjust the stimulator settings so that the frequency is 10
Hz and the number of pulses is 100 (10 seconds of pulses).
ligature
Figure 4: A ligature (thread) around the AV groove, and two ligature release
threads inserted BEFORE the ligature is gradually tightened.
4 Click Start, and then AutoScale. Type “Normal” on the
comment line. Press the Enter key. Record contractions for
about 15 seconds. Click Stop to halt recording.
5 Slowly tighten the knot a bit, making sure that the thread
stays in the AV groove. Then repeat Step 3, typing “Ligature
#1” on the comment line.
6 Continue to tighten and record for 15 seconds several
more time, labeling the traces “Ligature #2”, #3, etc.
Examine the recording each time. If the atria and ventricle
still contract in a coordinated fashion, but the time between
the arial and ventricular contractions are farther apart, this is
a first-degree block. If there are several (2-6) atrial
contractions between each ventricular contraction, in a very
regular pattern, this is a second-degree block (Figure 5). A
third-degree block is when the ventricle stops beating
completely, or its beats are far apart and not synchronized
with any atrial contraction.
7 Select Save in the File menu, but do NOT remove the
ligature until completing the next exercise.
Exercise 9: Independent Contractility
Aim: To determine which portions of the heart are capable of
beating on their own (have cells with pacemaker potentials).
Procedure
1 Remove the heart pin, wash it carefully, and get it back to
your instructor. It’s very easy to loose track of these, and then
someone gets stuck or the pin gets lost.
2 Carefully cut the attached blood vessels so that you can
remove the entire heart and put it in a Petri dish with Ringers.
3 Determine the heart rate of the atria and the ventricle
(beats/min.).
4 Carefully separate the three chambers of the heart in the
Petri dish. Determine the heart rate of each.
Data Analysis
Temperature and Drugs
In these sections of the experiment, you recorded cardiac
activity before and after the application of the test solution. To
extract the pertinent information from the data set, you should:
1 Scroll to the section of data that is relevant to the solution
being tested. Click AutoScale to maximize the size of the
response on the window.
2 Scroll to the Mark indicating the point when the application
of the solution was made. The GoTo command in the Marks
window can be used to locate the marks in the recording.
Figure 5: A ligature causes the atria (smaller peaks) and ventricle to
contract independently.
3 Measure the heart rate and the contraction amplitude at 30,
20 and 10 seconds before the mark (the control values), and at
the mark and every 10 seconds after the mark, for the duration
of the treatment (the experimental values).
Exercise 8: Threshold
4 At each time point, use the Display Time (Half or Double)
icons in the LabScribe toolbar to adjust the Main window, so
that a couple of contractions appear.
Aim: To determine whether the contraction of the heart
(ventricle) is graded or all-or-none.
Procedure
1 With the ligature still in place, use the stimulator to shock
the ventricle with a single pulse (still at 4V). If the ventricle is
not contracting at all, you can do this at any time, but if it is
contracting on its own occasionally, stimulate it between
these. Any contraction that occurs other than when you
stimulate should be ignored. You can “mark” this first
stimulation “4V”.
2 Reduce the voltage and repeat this procedure, marking
with the appropriate voltage, until you do not get a
contraction (i.e., the voltage is below the heart’s “threshold”).
5 Click the 2-Cursor icon (Figure 6), so that two blue vertical
lines appear over the Muscle channel on the Main window.
Move the cursors to positions on the recording window to
measure the following parameters:
• The amplitude (= force) of the heart contraction, which is the
voltage difference between the cursors placed on the baseline
and the peak of the ventricular contraction (Figure 7). The
amplitude is the value for V2-V1, displayed in the upper right
corner of the channel window.
• The period of one heart cycle, which is time difference
between cursors placed on adjacent peaks of ventricular
contractions. The period is the value for T2-T1, displayed on
the upper left side of the Main window.
3 Examine your record and decide if the heart’s contraction
is a graded or an all-or-none event.
4 Now, use the ligature-release threads to remove the
ligature, and wait until you get the heart back to normal
beating (as much as possible) before proceeding to the last
exercise. You may need to poke it (another form of
“stimulation”) regularly for a few minutes, and/or add some
epinephrine to help it return to a normal rhythm again.
Figure 6: The LabScribe toolbar.
considering the initial amount of stretch to be zero (0mm), and
each subsequent heart stretch to be 2 mm more.
3 Graph this force (y-axis) for each length (X-axis).
Ligature
1 Scroll to the appropriate section of data, and first go to the
initial 15 seconds before the ligature was tightened (first
degree block).
Figure 7: Using two cursors to measure the amplitude of a contraction.
6 Data can be entered in the Journal, by clicking on the
Journal icon in the LabScribe toolbar, and typing the values
for the amplitudes and periods and corresponding times in
the Journal window.
7 Convert heart cycle periods to heart rates by using the
following equation:
60 / one heart cycle period = heart rate
(sec/min)
(sec/beat)
(beats/min)
8 Graph heart rates and contraction amplitudes as a function
of time before and after the application of the solution, for
each experiment.
Refractory Period of the Heart
1 Scroll to the appropriate section of data.
2 Click the 2-Cursor icon (Figure 6), so that two blue vertical
lines appear over the Muscle channel on the Main window.
Move the cursors to positions on the recording window to
measure the period of time from the beginning of an atrial
contraction to the beginning of its ventricular contraction. The
period is the value for T2-T1, displayed on the upper left side
of the Main window.
3 Repeat this measurement in each 15 second recording to
see if/when a first-degree heart block occurs.
4 Visually inspect the rest of the 15 second recording to look
for second-degree blocks (Figure 5), and third-degree blocks.
Thought Questions
1 What are the effects of cold and warm Ringer's solution on
the rate and the amplitude of the ventricular contraction? What
Mechanisms are involved in producing these effects?
2 How do Acetylcholine and Epinephrine produce their effects
on the heart rate? On the amplitude of ventricle contraction?
2 Find the first extrasystole (Figure 3).
3 What effect does Atropine have on the heart? How does
Atropine work?
3 Click the 2-Cursor icon (Figure 6), so that two blue vertical
lines appear over the Muscle channel on the Main window.
4 When, in the cardiac cycle, does the refractory period for the
ventricle occur?
4 Place one cursor at the beginning of a naturally occurring
AP and at the location of the stimulus that created the
extrasystole. Record the time difference, T2-T1, between
these dips.
5 What is the significance of the long refractory period to the
function of the heart? What kind of action potential is this?
5 Examine the complete record of the refractory period
exercise. For all additional extrasystoles, make the same
measurement. The shortest time between a normal
contraction and an extrasystole should be close to the
refractory period of the ventricle.
7 How does the force of ventricular contraction change as it is
stretched? How does this compare with skeletal muscle?
6 Now look at all the contractions that did NOT have an
extrasystole. If a stimulus occurred during a contraction, and
a extrasystole was not produced, record the time.
7 If the heart rate was not the same as the stimulation
frequency, it can be assumed that the pulses were applied
at different times during the heart beat cycle. Examine the
complete refractory recording and determine:
• During which phases of the cardiac contraction cycle were
extra ventricular contractions recorded?
• During which phases of the cardiac contraction cycle were
extra ventricular contractions not recorded?
Stretching the Heart
1 Scroll to the appropriate section of data.
2 For each level of stretching, determine the force of
contraction (amplitude). Quantify the stretch on the heart by
6 Does the heart go into tetanus like a skeletal muscle?
8 In the ligatured heart in third-degree block, or when the
chambers are physically separated, the atria and ventricle
should beat at their own rates. Which rate is closest to the
heart rate seen in the turtle’s normal heart beat? Where are
the pacemakers for the atrial and ventricular rhythms?