Download Lab - Zoology, UBC

HEART FUNCTION IN THE FROG
(Martini 682-686, 687-692, 697-714))
Work in groups of 4
Objectives
 Describe the structure of the frog heart, and the events of the cardiac cycle in the frog; compare with
human
 Outline the conducting system of the heart in human and frog and relate to heart function
 Describe and explain the pacemaker of the heart
 State and account for the relative duration of each phase in the cardiac cycle
 Define and explain: refractory period, extra systole, compensatory pause
 Describe and explain the effects of autonomic drugs (acetylcholine, atropine, epinephrine) on frog
heart beat
SAFETY
 Students handling the frog must wear gloves.
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At the end of the lab:
Discard the frogs in the heavy black plastic bags labelled "biological waste"
Wash your dissecting instruments with soap and water.
Wipe your bench.
Wash your hands.
Equipment:
 double-pithed frogs
 diagrams and/or models of frog heart
 frog board or tray, pins
 Ringer's solution (13g of NaCl, 0.28g of KCl, 0.3g of CaCl22H2O, 0.4g of NaHCO3 for 2L of distilled
water) at room temperature.
 apparatus for recording heartbeat: recorder with 2 pens, amplifier, timer, transducer,
 thread, plasticine
 stimulator
 acetylcholine, 2 g/100 ml
 atropine sulfate, 1 g/100 ml.
 epinephrine solution, 1 g/100 ml.
 brown glass bottles with eyedropper (for pharmacological solution)
 watch glass
 timer
 Figures 1, 2 & 3.
Muscle cells of the heart contract as they perform their function: pumping blood. There are a number of
ways to examine this activity. One way is to look at the rate and strength of contraction in situ by
monitoring the actual movements of the ventricle itself. Another is to look at the electrical activity
generated by the heart each time it beats (ElectroCardioGram). Lastly, one can observe the effects of
heartbeat in the body by monitoring the pulse and blood pressure.
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In this lab, you will monitor the rate and the strength of contraction of a frog heart in situ.
I. STRUCTURE OF THE FROG HEART
Examine Figure 1 and familiarize yourself with the structure of the frog heart, and the vessels entering and
leaving it. Locate the single ventricle and the two atria. Blood enters the right atrium from the sinus
venosus, a thin-walled chamber on the dorsal side of the heart. The sinus venosus receives a mixture of
deoxygenated blood from the body and oxygenated blood from the skin and mouth. Oxygenated blood
from the lungs enters the left atrium. Both atria pump blood into the single muscular ventricle.
Ventricular contraction forces blood into the tubular truncus arteriosus which divides into left and right
branches at the anterior border of the heart. Each of these branches divides into three branches to (l) the
head, (2) the body and (3) the skin, mouth and lungs. Oxygenation takes place in the latter three organs.
Total mixing of blood from the two atria does not occur in the ventricle. A spiral valve in the truncus
arteriosus maintains separation so that oxygenated blood from the lungs is prevented from entering the
pulmocutaneous artery to the lungs, skin and mouth. The latter receives only mixed blood from the right
atrium, whereas the branches to the head and body carry both oxygenated blood from the left atrium and
mixed blood from the right atrium. Details of extent of mixing and mechanism of separation of the two
streams are still controversial.
Frogs are "cold-blooded". Their body temperature fluctuates with that of the environment. Hence they
expend less energy, and therefore utilize less oxygen than "warm-blooded" animals which must expend
energy to maintain body temperature. As a result inactive frogs can exist indefinitely without using their
lungs to breathe. They can pick up enough oxygen from the skin surface which is well-vascularized. Many
frogs spend the entire winter without breathing, buried in mud at the bottom of a pond.
The conducting system of the frog heart is essentially similar to that of the human, except that the
pacemaker of the frog is located in the wall of the sinus venosus.
NOTE: The entire central nervous system of the frogs used in this experiment has been destroyed by
pithing. As a result they cannot feel pain or respond to stimuli. But their tissues remain alive because the
heart continues to beat, distributing blood with oxygen and nutrients to them.
Perform Exercise A before hooking the frog heart to the transducer (Exercises B, C, D).
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Figure 1: Anatomy of the frog's heart: (a) ventral view showing the single truncus arteriosus leaving the
undivided ventricle; (b) longitudinal section showing the two atrial and single ventricular chambers; (c)
dorsal view showing the sinus venosus (pacemaker).
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II. OBSERVATION OF THE CARDIAC CYCLE
EXERCISE A
Watch a demonstration of the technique required to expose the frog's heart.
Obtain a double-pithed frog and fasten it on its back in the tray provided. Lift the skin over the abdomen
and cut through it with scissors in the mid ventral line from the lower end of the abdomen to the lower jaw.
Then cut through the body wall a little to one side of the mid-line. Cut through the shoulder girdle. Be
careful to avoid the vessels entering and leaving the heart. Carefully open the pericardium and expose the
beating heart. Keep the heart moist at all times with Ringer's solution applied with an eyedropper.
Determine heart rate per minute (count ventricular contractions for 30 seconds and multiply by 2).
Heart rate =
Observe the sequence of contractions of the frog heart. If the heart is beating too quickly to observe the
sequence of events, cool the heart with ice or cold Ringers solution.
Identify the sinus venosus (tip the ventricle forward and look on its dorsal surface), left and right atria,
ventricle, truncus arteriosus. Observe carefully. Do all parts of the heart beat at the same rate?
What is the sequence in which these parts contract?
Describe the changes in the ventricle during systole and diastole. Observe its size, and shape.
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III. RECORDING THE CARDIAC CYCLE
EXERCISE B
Attach the thread to the transducer lever with plasticine. Arrange the bath so that the thread is vertical. Do
not pull the thread too tight; it should be taut, but should not stretch the heart excessively. Balance the
lever with plasticine as demonstrated. Keep the heart moist at all times with Ringer's solution.
Adjust the gain and offset controls on the amplifier so that a deflection of at least 2.0 cm is obtained with
each ventricular contraction.
Record a series of at least l0 contractions with the chart running at moderately high speed (note speed).
You should be able to obtain a trace similar to that shown in Figure 2. Ventricular contractions should be
spaced at intervals of not less than 2 cm. Identify atrial and ventricular contractions on the chart. Atrial
contraction precedes ventricular contraction and is much weaker. If your record is unclear try adjusting the
set-up. The tension on the thread attaching the heart to the transducer lever is often at fault.
Make sure that you have a clean trace showing 3 contractions. You will have to include them or a
photocopy of them in your lab report. Label the chart clearly, and record chart speed and direction. Be sure
that you can identify which part of the trace results from contraction, and which from relaxation; label
before you dismantle the apparatus.
Figure 2: Cardiac cycle of frog's heart using one transducer attached
to the ventricle (1) and using two transducers (2) one recording only
the atrium contraction (top) the other recording only the ventricle
contraction (bottom).
You should obtain a trace similar to (1). As you can see on this figure,
this trace is a combination of the contractions of the atria and the
ventricle.
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IV. THE REFRACTORY PERIOD AND EXTRA SYSTOLE
EXERCISE C
Be sure that the event-marker pen (which records electrical stimuli) is exactly aligned with the amplifier
pen (which records contraction). Set the speed of the chart recorder at 2.5 mm/sec.
Set the stimulator:
Start with a stimulus intensity of 10-20 volts (test the stimulator for muscle contraction of the frog by
gently touching the electrode to the leg – if a muscle twitch is not observed, increase the voltage) and the
frequency of stimulation should be set at 100 stimuli/sec.
Set the stimulus frequency at 100 stimuli/sec.
Before doing the stimulation, the heart should be slowed down with ice or cold ringer (this will make it
easier for you to stimulate the ventricle precisely during the systole or the diastole as you wish it). Start the
chart running. Switch on the stimulator to the repeat mode and touch the ventricle at different phases of its
cycles. This will require a number of tests - repeat until you have records of stimuli that coincide with all
phases of the cardiac cycle. Watch for the appearance of extra systoles - beats induced by the electrical
stimulus (Figure 3). Your objective is to determine when, during the cycle, the heart is refractory (i.e.
when the electrical stimulus does not result in an extra contraction).
Labelled chart records (or copies of them) of this experiment will be included in your lab report. Indicate
stimuli that failed to induce contractions as well as those that induced extra systoles. Correlate these with
stages in the cardiac cycle. Note whether the pause between beats (diastole) is of normal duration after an
extra systole (premature contraction) is induced. Measure intervals for several normal beats and compare
with that after a premature contraction.
Figure 3: Refractory period and compensatory pause of the frog heart when stimulated at various times
during the cardiac cycle.
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V. EFFECTS OF AUTONOMIC DRUGS
EXERCISE D
Record a series of l2 control heartbeats on a chart moving at moderate speed (2.5 mm/sec), then bathe the
heart with epinephrine solution using an eyedropper. Start with five drops. Indicate on the chart recording
the time of epinephrine addition and the number of drops added. Continue recording until you notice an
effect. If no effect is observed within 3 to 5 minutes, apply five more drops of epinephrine. When an
effect has been recorded, rinse the heart thoroughly with Ringer's solution.
When the heart has returned close to its normal rate, again record a second sequence of control heartbeats
(most likely this rate will be higher than the first control). Using a different eyedropper, bathe the heart
with 5 drops of acetylcholine solution, noting the time on the chart. Continue recording until an effect is
observed. If no effect is observed within 3 to 5 minutes, add more acetylcholine.
When a definite effect has been obtained, or if the heart stops beating, bathe the heart freely with atropine
sulfate, indicating the time of atropine addition on the chart. Continue recording until recovery is observed.
Wash copiously with Ringer's solution.
Be sure to label all activities on your chart as you proceed. You will include labeled charts (or copies of
them) in your lab report.
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Calculate the following heart rates:
Control heart rate and highest heart rate reached with epinephrine
Second control heart rate, lowest value reached with acetylcholine, highest value reached with atropine.
Record your results in Table 2.
To calculate heart rate:
(1) Measure out the distance (in mm) for 10-12 complete heart beats on the chart recording = heart beat
interval in
# of beats/x mm.
(2) Use the following equation to calculate heart rate:
HR = heart rate interval (# of beats/x mm) X chart speed (x mm/sec) X time conversion factor
(60sec/min).
Table 2: Effects of autonomic drugs on cardiac cycles.
Control
Epinephrine
HR (beats/min)
New Control
Acetylcholine
Atropine
Note: Atropine is a competitive inhibitor of acetylcholine. It acts at receptor sites in smooth and cardiac
muscle, but not in skeletal muscle. It is not produced by the body, but is sometimes used as a drug to
inhibit the effects of the parasympathetic nerves.
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VI. AUTOMATICITY OF THE HEART
EXERCISE E
Using the pin and string, lift the ventricle, carefully cut through the arteries and veins and remove the heart.
Be careful to keep the sinus venosus intact; cut the veins, which drain into it. Place the heart in a watchglass filled with Ringer's solution. Observe. Count the beating rate after one minute.
Using scissors carefully cut through the junction between the sinus venosus and the right atrium. Wait
several minutes. Both parts of the heart should continue to beat. Count the rate for each part.
Cut the atria free from the ventricle. Describe the activity of each segment. Count the rate (if present) for
each segment. Observe for l0 minutes. If the ventricle still shows no activity, stimulate it by pricking it
with a pin. Determine its rate.
Record your results in Table 3 and tabulate heart rate for each chamber under each condition.
Note: This procedure is not always successful. If your frog's heart fails to beat after separating the atria
from the ventricle, observe and describe one that does or predict what you would expect to see.
Table 3: Automaticity of the heart.
EXPERIMENTAL
PART OF THE HEART
CONDITIONS
UNDER OBSERVATION
Isolated heart
Whole heart
Section between the sinus
Sinus venosus
venosus and the right atrium
Rest of the heart
Section between the atria and
Atria
the ventricle
Ventricles
RATE OF BEATING
(beats/min or predicted rate)
CLEAN UP:
Turn off and disconnect electronic equipment.
Discard the frogs in the heavy black plastic bags labeled "biological waste"
Wash glassware, rinse it thoroughly with water, and leave it to dry on paper towels on your bench.
Rinse the frog board with water. DO NOT USE DETERGENT OR BLEACH.
Wash your dissecting instruments with soap and water.
Wipe your bench.
Leave your space clean and tidy.
Wash your hands.
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NAME & section #:
Biology 153 LAB REPORT (2002-2003)
EXERCISE 3: HEART FUNCTION IN THE FROG.
Do not copy word-for-word from text.
INTRODUCTION
State the purpose of this exercise
MATERIALS AND METHODS
See the lab manual.
RESULTS
Use separate pages.
A. OBSERVATION OF THE CARDIAC CYCLE (Exercise A)
- Record observations as instructed in the lab manual. Answers to questions should be brief.
B. RECORDING THE CARDIAC CYCLE (Exercise B)
- Include fully labelled (atrial systole, ventricular systole, ventricular diastole, chart speed) section of chart
paper from the recorder with your report.
- In a table show the following calculations:
a) heart rate in beats/min.
b) duration of the atrial systole
c) duration of the ventricular systole
d) duration of the ventricular diastole.
C. THE REFRACTORY PERIOD AND EXTRA SYSTOLE (Exercise C)
- Include fully labelled chart recordings showing the effects of stimulation at different points of the cardiac
cycle. Include:
a "normal" recording for comparison.
a recording showing the extra systole and compensatory pause. Indicate where during the cardiac cycle the
stimulus would be applied to produce the extra systole.
Label chart speed and pen direction
- In no more than five lines describe what you observed.
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D. EFFECTS OF AUTONOMIC DRUGS (Exercise D)
- Include a fully labelled chart recording showing the effects of epinephrine, acetylcholine and atropine
sulfate. Include:
a recording of the control heart beats and a recording following drug application until a change in rate or
contractility can be seen (indicate where the drug was added).
cut down the tracing if the effects take too long and note the time (do not fold tracings to fit the page).
- In a table show the changes in heart rate before and after drug application.
- Illustrate with a bar graph the effect of these autonomic drugs on the heart rate.
- In five lines describe your results.
E. AUTOMATICITY OF THE HEART (Exercise E)
- Tabulate your results.
- In 5 lines describe your results. If you are missing data, include a column for predicted results.
DISCUSSION
Use spaces provided below. Do not use extra pages.
THE REFRACTORY PERIOD AND EXTRA SYSTOLE (Exercise D)
1) When during the cardiac cycle must the ventricle be stimulated in order to produce an extra systole?
Explain why you obtained different responses depending on when you stimulated the ventricle? (some
stimulations will cause an extra-systole, others will not)
2) What is the physiological explanation for the compensatory pause?
3) What is the advantage for cardiac muscle to exhibit an absolute refractory period that is nearly as long as
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its contraction?
4) What is the advantage for skeletal muscle to exhibit a refractory period, which is so small as to be
negligible?
EFFECTS OF AUTONOMIC DRUGS (Exercise D)
In terms of the neural regulation of the heart, explain the effects of epinephrine, acetylcholine and atropine
on heart rate. What is the effect on heart rate and contractility and how do the drugs act to produce these
effects?
AUTOMATICITY OF THE HEART (Exercise E)
1) Explain why the sinus venosus, the atria and the ventricles beat with different rates when they are
separated from each other.
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2) Will the rate of beating of the whole heart be similar to the rate of the isolated ventricle, of the isolated
atria or of the isolated sinus venosus? Why?
The End
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