Biology 450 - Animal Physiology
Fall 2007
Lab 8 – Cardiovascular Physiology of the Frog
In this lab, you will expose a frog heart in situ in order to observe the cardiac cycle
in an active heart, investigate the regulatory effects of two neurotransmitters on
heart rate and contractile strength, and attempt to identify the nature of two
unknown compounds through their effects on the frog heart. In addition, you will
look for a relationship between the stretching of the ventricle and the force of
contraction (Starling’s “Law of the Heart”).
Background
Cardiac cycle
Individual heart muscle cells, if isolated in saline solution, will contract
spontaneously at a fairly regular rate. Spontaneous contraction in muscle is
sometimes referred to as myogenic activation, and regular myogenic activity is
referred to as “autorhythmicity.” A specialized bundle of tissue, known as the
“pacemaker” (usually the sinoatrial, or SA, node), is responsible for setting the
heartbeat rate since it fires inherently faster than the spontaneous rate of other
heart cells. The activity of the SA node ultimately stimulates the contractile cells of
the myocardium.
When the cardiac muscle fibers in the atria or ventricles are stimulated, contraction
occurs. Since the cells are united by gap junctions, the excitation producing
contraction is spread from cell to cell and the muscle contracts as a single unit,
ejecting blood from the chamber. After contraction, the heart chambers relax and
refill. A complete heart beat or cardiac cycle consists of atrial contraction and
relaxation followed by ventricular contraction and relaxation. These ventricular
events are typically referred to as systole and diastole. The atria are normally
contracted for about 0.1 seconds and relaxed for 0.7 seconds. Ventricular systole
lasts for about 0.3 seconds with diastole taking 0.5 seconds. A single cardiac cycle
thus takes about 0.8 seconds when the heart rate is 75 beats per minute. The lag
time between the contraction of the atria and ventricles results from a delay
imposed on the conduction system at the atrioventricular node.
One additional aspect of the cardiac cycle to consider is ventricular ejection, which
occurs when ventricular pressure exceeds that within the arteries and blood is
ejected from the heart. The sudden increase in volume and pressure in the arteries
causes them to expand elastically; they decrease in size more slowly during
diastole.
In today's lab you will be examining the cardiac cycle of the frog, Rana catesbeiana,
and measuring how certain parameters affect it. This organism is an amphibian
(obviously), which means that its heart is three-chambered rather than fourchambered like an avian or mammalian heart. It has a single ventricle from which
all blood exits. The large dorsal sinus venosus is the location of the pacemaker.
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Regulation of heart activity
The control of cardiac contractility involves the activity of both intrinsic (within the
heart) and extrinsic (from outside the heart) factors. The pacemaker cells act as an
intrinsic control of heart rate, since an excised heart will still beat at a regular rate.
Extrinsic controls include factors such as nervous and hormonal inputs, which can
influence both heart rate and cardiac output.
With regard to nervous control of the heart, the primary regulating center is located
in the medulla oblongata of the brain, and the heart is innervated by both
sympathetic and parasympathetic nerve fibers that terminate at the SA node. The
neurotransmitters released by these nerves affect both heart rate (chronotropic
effects; chronos = time) and the strength of contraction (inotropic effects), by
influencing the timing and magnitude of ion currents across the cell membrane.
The vagus nerves contain parasympathetic cholinergic neurons that release
acetylcholine at their terminals. This neurotransmitter binds with muscarinic
receptors and initiates many effects, including a cascade that increases the number
of K+ channels in the open position; thus keeping the membrane near the
equilibrium potential for K+ and making depolarization more difficult.
The sympathetic cardiac nerves contain adrenergic neurons that release
epinephrine or norepinephrine, depending on the species. (Amphibians release
adrenergic
receptors at both the SA node and in the myocardium. This binding can cause a
variety of effects, including an increased inward flux of Ca++ into the cell.
The function of the muscarinic and adrenergic receptors in the heart can be
revealed by examining how the heart responds when these receptors are activated
or inactivated. Compounds that activate a receptor are called agonists, while
compounds that inactivate a receptors by binding to it are called antagonists. Most
medications used in the treatment of heart disorders are antagonists for either
muscarinic or -adrenergic receptors.
In this lab you will examine the chronotropic and inotropic effects of two agonists,
epinephrine and acetylcholine. You will also quantify the effects of an unknown
antagonist and, based on your observations, identify the type of receptor to which
it binds.
Lab Procedures
You will use a 10g force transducer in combination with Chart to measure heart rate
and contractility. Although the frog heart is quite hardy, you must avoid damaging
it unnecessarily and keep it wetted during the course of your experiments.
Setup
The hardware setup for these experiments is fairly simple.

Arrange the force transducer so that it is 10-15 cm off the benchtop with the
hooks facing towards you (in other words, to record a horizontal rather than
a vertical force).
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
Make the appropriate connections to the bridge amp and the PowerLab, then
start Chart and make sure all hardware and software settings are
appropriate.
Dissection
You need to expose the heart of a frog in
order to measure its activity. You will be
supplied with a pithed or decapitated
frog by your instructor.
During the dissection, be very careful
not to damage the heart or its major
vessels. Throughout your experiments,
keep the heart and internal organs wet
with frog Ringer’s.
1. Place the frog in dissecting pan
and use dissection pins to hold the
frog in place.
Ventral view
2. Using a scalpel or sharp scissors,
make a lateral incision through
the skin across the chest of the
frog (from armpit to armpit). The
first incision should expose the
muscle and connective tissue.
Carefully cut through this layer of
tissue to expose the heart. Gently
remove the pericardium from
around the heart. The frog heart
has a single ventricle, which
pumps blood to both the lungs
and the rest of the body. Remove
other tissue as necessary to get a
clear view of the heart.
Dorsal view
3. Using forceps, insert a fishing
hook into the apex of the heart,
being careful not to puncture the
chamber of the ventricle.
4. Attach the line at the other end of
the fishhook to the force
transducer, then gently move the
frog away from the transducer so
the heart generates slight tension
on the line. The transducer
should be low enough so that the
heart pulls against it primarily in
the horizontal plane. When
properly positioned, the voltage
output of the force transducer will
be proportional to the force of
Internal view (ventral)
Anatomy of the frog heart
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contraction of the heart without overstretching the heart. Adjust the voltage
range in Chart to maximize the visibility of the heartbeat.
Exercise 1 – Observation of the cardiac cycle
Although not an experiment per se, you may find it interesting to observe the
cardiac cycle in the frog heart. You can see the relative timing and force of atrial
and ventricular contraction, and it is usually possible to perceive the blood moving
through the chambers and blood vessels. You should also be able to observe the
expansion of the major arteries during systole.With careful adjustment, both atrial
and ventricular contractions may register on the force transducer.
Exercise 2 – Starling’s “Law of the Heart”
Starling observed that increased filling of the ventricle leads to more forceful
contractions, and deduced that this was because of the increased stretching of the
muscles prior to contraction. In this exercise, you will apply increasing tension to
the ventricle, thus stretching it more and more, and examine the resulting force
production of the muscle.
1. Adjust the position of the frog prep so that the tension on the line is just
sufficient to provide reliable readings from the force transducer. Begin
recording in Chart.
2. Slowly move the prep further from the force transducer, watching the force
production. (Note that the baseline reading will increase as tension increases.
You are interested in the additional force generated by the ventricle.)
Continue increasing tension until force production begins to decrease or there
is danger of damaging the prep.
3. Return the prep to its original configuration.
Exercise 3 – Effects of known agonists
In this exercise you will examine the response of the heart’s activity to the
cholinergic agonist acetylcholine, and to the adrenergic agonist epinephrine.
Obtain small amounts of the epinephrine and acetylcholine solutions, and be sure
your setup is still healthy. Keep the heart wetted with Ringer’s solution.
1. Record the activity of the heart for one minute to provide baseline data.
2. While still recording, drip epinephrine solution onto the heart at about one
drip per second for about 10 seconds. Use the comment feature of Chart to
mark your recording. Watch for changes in heart activity over the next five
to ten minutes. If you are not seeing any changes, try repeating the dose.
3. When the heart’s activity no longer appears to be changing in response to
the epinephrine, rinse the heart and surrounding areas well with Ringer’s,
then wait five minutes to make sure the epinephrine is no longer having an
effect. You can stop the recording to collect data on the epinephrine at this
point.
4. Repeat steps 2-4 for acetylcholine.
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Exercise 4 – Effects of an unknown antagonist
In this exercise you will examine the effect of an antagonist on the activity of the
heart and on the effects of your two agonists. Often, antagonists block the effects
of agonists on a particular class of receptor.
Again, record the activity of the heart for one minute to provide baseline data.
1. Drip the antagonist onto the heart and watch for any changes in heart
activity over the next five to ten minutes.
2. Do not rinse the heart, but instead apply one of the agonists from exercise 2.
Compare the response to that seen earlier.
3. Rinse the heart well with Ringer’s
4. Repeat steps 2-4 for the other agonist.
Exercise 5 – Effects of an additional agonist
The heart has additional classes of receptors in addition to the cholinergic and
adrenergic ones. In this experiment, you will examine the effects of another
neurotransmitter, serotonin, on heart function. To do this, follow the protocol used
for the agonists in exercise 2.
Additional experiments
If time allows, you may want to try additional experiments, such as obtaining an
EKG from the frog, or blocking the pacemaker signals between the atria and
ventricle by applying a ligature. Ask your instructor for assistance if you get this
far.
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