Frog Heart Lab
Fall 2006
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CARDIAC MUSCLE, THE FROG HEART
PURPOSE:
1.
2.
3.
To examine the basis of the heart rhythmicity.
To examine factors that influence contraction force.
To examine factors that influence of various regulatory factors heart rate
Introduction
In these exercises you will explore the basis of modulation of cardiac activity. You will examine the effects of
stretch changes in internal environment and neural/endocrine modulators on the ability of the heart to do work.
Although the frog heart (chosen in large part b/c the frogs are an R-selected species, at least compared to the
turtles that we used to use) has been chosen for these experiments, the principles examined in his laboratory
apply to mammalian hearts as well.
Physiology Background
Cardiac muscle has an inherent rhythmicity that is modified - sped up or slowed down - by neural and hormonal
factors. The basis of this heart beat is the spontaneous depolarization of pacemakers cells that essentially bring
themselves to threshold thereby producing an action potential that travels through the cardiac muscle and
stimulates its contraction. Specifically, the cardiac action potential travels through (a) a specialized conduction
system of modified cardiomyocytes –that is, the Bundle of His and the Purkinje Fibers - that brings the signal
into close proximity to each cardiomyocyte and (B) from any given cardiomyocyte to adjacent cardiomyocytes
through gap junctions. Thus the cardiac cells will contract as one group following the depolarization of the most
frequently depolarizing group of pacemaker cells (By the way, the fastest group is at the Sino-Atrial Node or SA
node and the Second fastest is at the Atrio-Ventricular Node or AV node).
At cardiomyocytes, the action potential opens calcium channels increasing cytoplasmic calcium concentrations.
This increased cytosolic Calcium then opens Calcium channels on the Sarcoplasmic Reticulum; leading to
additional Calcium release into the cytosol (the process is termed Calcium-Induced Calcium Release). As with
skeletal muscle, cytosolic Calcium binds to troponin which lifts tropomyosin off of actin thereby permitting the
phosphorylated myosin crossbridges to bind to actin and the slide filament activity to occur.
Like neural tissue, cardiac tissue is observed to have an "all or none" activity. The population of cardiac cells
will pump blood (contract) or it will not contract (relaxation).
The heart will contract with more force if more work is demanded of it. This is advantageous, since the cardiac
efficiency is based on the amount of blood moved. If more blood enters the heart, more work is necessary. By
contracting with more force the heart rises to the work challenge with each contraction. This qualitative
relationship is somewhat quantified via Starlings law of the heart, essentially analogous to the length tension
relationship observed in skeletal muscle. That is, an increase in end diastolic volume of the heart (= increased
ventricular stretch) increases the force of cardiac contraction by bringing the ventricular myocytes closer to their
“optimal length”. Of course this means that more blood will be pumped per stroke and thus per minute,
according to the relationship: Cardiac Output (CO) = Heart Rate (HR) X Stroke Volume (SV).
Although the heart contracts with a periodicity determined by pacemaker cells; hormones and neurotransmitters
can speed up or slow down this rhythm. The right vagus sends mostly parasympathetic nerves to the heart.
Thus, stimulating the vagus slows the heart down. In contrast, adding epinephrine increases the heart rate.
Later in these exercises you will be asked to consider and test the effects of ACh, and Adrenaline, as well as
ACh-receptor agonists (pilocarpine) and antagonists (atropine) on cardiac function.
PROCEDURE
No Scalpels. All cutting should be done with scissors! You will be observing cardiac function as a contraction
rate and force of contraction (Amplitude height). Cardiac function is dependent on moving fluid, so the more
blood remaining, the better your preparation will work.
1. Select a pithed frog and pin it out, ventral surface up in a dissecting tray using large dissecting pins
through the “ankles” and “wrists”. Be sure the limbs are fairly taught to prevent involuntary movement.
2. Next, dissect the frog according to the directions in fig 1, below.
3. Attach the apex of the heart to the force transducer as diagrammed in figs 1 & 2.
Frog Heart Lab
Fall 2006
Figure 1. Dissection of the frog to expose the heart.
Figure 2: Arrangement of the heart and force
transducer. Note the relatively Horizontal orientation
of the thread linking the heart and the transducer.
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Frog Heart Lab
Fall 2006
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Recordings
1. At this point in the semester you should be fairly familiar with the PowerLab system. Open Chart, set
the channel one speed to a fairly slow rate, and zero the force transducer recording. (remember you can
do this in the bridge amp dialogue box for the first recording channel).
2. After you get a stable trace, record the heartbeat under baseline conditions for 2 to 5 minutes. Make
sure to get at least a dozen contractions recorded.
3. Cardiac refractory period and summation:
a. Place the stimulating electrodes on the atria and apply 1 ms-long stimuli to try and evoke a
second contraction of the heart during the valley between contractions. This period represents
the latter part of ventricular relaxation or diastole. Try increasing the voltage up to 2V to get an
“extra” contraction. If unsuccessful, try shifting your electrodes around rather than increasing
the voltage more.
b. Using a stimulus approximately 25% stronger than that used to evoke the initial 2nd contraction,
try to evoke a second contraction at various points during the following portions of the cardiac
cycle: Beginning of systole, Systole midpoint, Systole peak, beginning of diastole, diastole
midpoint, diastole valley. Your TA will explain what portions of the recording correspond to
these values.
4. Vagal Stimulation: Locate the right vagus using the glass probes. The vagus is a whitish cord close to
the carotid artery and jugular vein. Do not cut these vessels. Stimulate the vagus with the PowerLab
at 1 V, 5 pps. You may have to increase the voltage. Continue the stimulation for 1 to 2 minutes.
5. Starling’s law of the Heart: Slowly increase the height of the force transducer by 2-3 mm per cardiac
cycle, thereby increasing the stretch on the heart. Record the change in contraction force with each
height change.
6. Effect of temperature on cardiac function:
a. Pool cold saline around the heart in the body cavity. Record the changes in frequency and
force you observe as the heart cools and re-warms.
b. Pool warm saline around the heart. Record the changes in frequency and force you observe as
the heart warms and re-cools.
7. Effects of drug treatments: Next examine the effects of various drug treatments. These effects should
be monitored as immediate, at 1 minute, and at 3 minutes following application. For each, record the
effects on heart beat frequency and strength. After each treatment, thoroughly wash the drug out with
repeated saline flushes.
a. Add 2 or 3 drops Nicotine to the heart.
b. Add 2 or 3 drops Acetylcholine to the heart.
c. Add 2 or 3 drops Epinephrine to the heart.
d. Add 2 or 3 drops Pilocarpine to the heart.
e. Add 2 or 3 drops Atropine to the heart; follow after 3 minutes with pilocarpine (without a saline
rinse in between).
8. Heart Dissection: Remove the heart from the body cavity, and place in a beaker of oxygenated saline.
Observe and record the pace of the heart beat in the beaker, then use your scissors to remove the atria
from the remainder of the heart. Record the effects of atria removal on the rate of the heartbeat.
Data and Questions
1. Record all of the data requested in the procedures above.
2. Baseline heart rate and contractility: Based on your baseline, what is the heart contraction time? What
is the heart rate?
3. Cardiac Refractory Period and Summation: Were you able to evoke a second cardiac contraction before
the first was complete, if you applied three pulses in a row were you able to achieve summation of each
contraction after the first? What do these data tell you about the refractory period of the heart muscle?
How does this parameter compare to that of skeletal muscle?
Frog Heart Lab
Fall 2006
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4. Vagal Stimulation: What happens to the frequency/force of the heart beat when you stimulated the
vagus nerve? Explain in terms of the neurotransmitter(s) being released and their affect on cardiac cells.
5. Starling’s Law of the Heart: Plot the relationship between heart stretch and the force with which it
contracted. What is the physiological basis of this phenomenon? What is its relevance?
6. Effect of Temperature on Cardiac Function: From a simple biochemical standpoint, briefly hypothesize
why cool and warm saline affected cardiac function in the manner they did.
7. Effects of drug treatments: What did application of the various drugs tell you about the types of
receptors present on the heart tissue and their roles in regulating cardiac function?
8. Heart Dissection: How did removal of the atria affect the frequency of the heartbeat? Explain this
phenomenon in terms of the various pacemakers in the heart.