Yixi Liu

Electrical and Mechanical Properties of the Frog

Heart
Yixi Liu
Group members : Gloria Kwak, Ramneet Grewal, Jessica Yee
NPB 101L Section 03
TA : Kathryn Dorhout
October 27, 2015

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INTRODUCTION
The heart is a significant organ in life because it is the power source of the circulatory
system. It is responsible for generating high pressure to allow the blood to flow to low
pressure regions. The heart drives sufficient blood flowing through the organs and tissues to
supply them with oxygen and nutrients and at the same time, the blood removes waste and
metabolites. The circulatory system is made up of pulmonary circulation and systemic
circulation. The pulmonary circulation consists of the lungs and skin capillaries; it transports
deoxygenated blood from the heart to the lungs through the pulmonary artery and carries
oxygenated blood back to the heart through the pulmonary veins. The systemic circulation
contains systemic capillaries which drive blood circulation throughout all the organs except
the lungs. The heart is located in the middle of the chest cavity and most vertebrate hearts
contain atria and ventricles. The atria are the receiving chambers located in the upper portion
of the heart and the ventricles, which are located in the lower portion of the heart, are used for
pumping blood to the body. Human hearts contain four chambers with a pair of atria and a
pair of ventricles. The systemic circulation drives O2-poor blood to the right atrium through
venae cavae. Then the blood flows to the right ventricle with the open right atrioventricular
valve, which also called the tricuspid valve, and is pumped out by the pulmonary artery to the
lungs. These atrioventricular (AV) valves are located between the atrium and the ventricle on
both sides of the heart and ensure the unidirectional flow of blood from the atrium to the
ventricle. After the purification in the lungs, O2-rich blood flows to the left atrium through the
pulmonary veins. Then the blood flows to the left ventricle with the open left atrioventricular
valve, which is also called the bicuspid valve or mitral valve. The left ventricle pumps the

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blood into the systemic circulation to different systemic organs throughout body such as the
brain, the digestive tract and kidneys through the aorta (Sherwood, 2010, p. 303-306).
The heart is made up of striated cardiac muscles and muscle fibers are interconnected
by intercalated discs (Sherwood, 2010, p. 308). Autorhythmic cells make up 1% of the
cardiac muscle cells, but they are very important because they can slowly depolarize to the
threshold and initiate action potential for muscle contraction. Action potential spreads via gap
junctions through the atria. Specialized autorhythmic cells in mammalian hearts are located at
four parts of the body: the sinoatrial node (SA node), the atrioventricular node (AV node), the
bundle of His (atrioventricular bundle) and purkinje fibers. The SA node is considered as the
pacemaker because it contains cells that have the fastest rate of generating action potential
(Sherwood, 2010, p. 309-311). There are five phases for cardiac muscles to generate an action
potential. Phase 0 is when fast Na+ channels open at the threshold, the rapid depolarization
and influx of sodium ions causes the rapid rising of action potential. Phase 1 is when
potassium ions repolarize and efflux with the opening of K+ channels; membrane potential
decreases to 0 mV. Phase 2 is a plateau while the slow influx of calcium ions travels through
L type Ca2+ channels coupled with K+ efflux. Phase 3 shows the repolarization and efflux of
K+ cause the rapid decrease in membrane potential to -90 mV. Phase 4 shows resting
membrane potential is maintained at -90 mV with the opening of K+ channels (Sherwood,
2010, p. 314-315).
Cardiac muscles, similar to skeletal muscles, have the excitation-contraction coupling
process. An action potential initiated in a cardiac contractile cell travels down T tubules.
Calcium ions enter the cytosol through L-typed Ca2+ channels and cause the rapid release of a

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large number of Ca2+ from the sarcoplasmic reticulum to the cytosol through ryanodine
Ca2+-release channels. This Ca2+ - induced Ca2+ release process results in a great increase in
cytosolic Ca2+ concentration. Then calcium ions bind to troponin. Similar in the skeletal
muscles, cross-bridge cycling occurs and leads to contraction (Sherwood, 2010, p. 315).
There are some differences between mammalian and amphibian hearts. For example, in
mammalian hearts, such as human hearts, there are two atria and two ventricles. The main
pacemaker is the SA node, and other autorhythmic cells are located at the AV node, the
bundle of His and purkinje fibers. However, in amphibian hearts, such as frogs, there are only
three chambers - two atria and one ventricle. The main pacemaker of the heart is the sinus
venosus, and frog heart does not contain the AV node. Instead of this, it contains a funnel of
cells between the atria and ventricle, and thick muscles of frog ventricle walls separate
oxygenated from deoxygenated blood, and still allow the delay between atria and ventricle
contraction. Rather than formal bundle of His or purkinje fibers, frog hearts contain electrical
pathways.
Systole is the period when the ventricle contracts and heart ejects blood, and diastole is
when the heart fills up with blood during ventricular relaxation. There are two types of
refractory periods. One is called absolute refractory period, which means no matter how large
the stimulus, no action potential could be generated. The other one is called relative
refractory period, which means another action potential could be generated with large enough
stimulation. Extrasystolic contraction is when the muscle cell is stimulated during the relative
refractory period, a second action potential can generate a second systolic contraction. A
compensatory pause is the period between extrasystolic contraction and the following

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ventricular contraction (Sathya Subramani, Priya Mammen, 2001, p. 511). Due to Frank
Starling Law of the Heart, the contraction after the compensatory pause would be larger.
Frank Starling Law of the Heart demonstrates that the force of the contraction of the heart
changes in response to venous return, which means greater filling of the ventricle will
increase end-diastolic volume, and lead to a greater stroke volume (Sherwood, 2010, p. 328).
The parasympathetic nervous system (PNS) and sympathetic nervous system (SNS)
make up the autonomic nervous system. The PNS slows pacemaker, decreases the heart rate
and cardiac output and weakens atrial contraction. The PNS acts via the vagus and releases
acetylcholine (Ach) to decrease the function of vagal activity, which would cause the
decrease of the heart rate and lead to bradycardia (Gary et al., 1992). The SNS increases the
pacemaker, increases the heart rate and cardiac output and increases strength of ventricular
contraction. The SNS acts via norepinephrine (NE) to generate greater contractions.
Vagal escape is the phenomenon that the heart beat can return after the heart stops with
continuous vagal stimulation because other pacemaker cells outside the SA node would take
over when SA node is over stimulated and cannot function properly (JH et al., 1988). The
presence of vagal escape is regulated by the PNS.
The subjects of this experiment were double-pithed frogs. The goals of this experiment
were, first, to investigate the electrical and mechanical activity of the frog heart. It was
expected to observe clear atrial and ventricular contractions and electrical activities, and
ventricular contraction force should be larger than atrial contraction. Second, by applying
stimulation during late diastole and early diastole, it was expected to observe extrasystolic
contractions after late diastole stimulus, and no extrasystolic contractions after early diastole

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stimulus. Third, by gradual increasing the stimulus voltage on the vagus nerve, it was
expected to observe the presence of bradycardia and cardiac arrest. Finally, by applying
epinephrine to the frog ventricle, the increase in the heart rate was expected.

MATERIAL & METHODS

Detailed procedures of this experiment were illustrated in the NPB 101L Systemic
Physiology Lab Manual (Erwin Bautista, Julia Korber, 2009, p. 44-53). For the preparations,
first, the skin of the frog was cut from lower abdomen to jaw, then the abdominal muscles
and the sternum were cut medially. The overlaying pericardium was removed after the
exposal of the heart. Then, the vagus nerve within the thoracic cavity was isolated. After
calibrating the transducer, the insulation of two sides of a wire were burn off and the
uninsulated region was inserted into the ventricle. The other side of wire was connected to the
force transducer. Ringers solution was used to keep the frog moist and the frog needed to
be firmly massaged on the legs and viscera, especially the liver, to increase venous return.
After all the preparations, the mechanical signals and electrical activities would be
recorded by a computer software - BioPac Student Lab, and it was expected to be observed
the signals and electrical activities of atrial contraction and ventricular contraction. However,
during the whole process of the experiment, electrical activities were not observed. Then in
order to find out the properties of extrasystolic extractions, different stimulus voltage during
late diastole and early diastole with constant duration (2.0 mS), delay (0.02 mS) and
frequency (20 pps) were applied. In the next part of the experiment, the vagus nerve was
stimulated directly by changing stimulus voltage to see the presence of bradycardia and
cardiac arrest, with constant duration (2.0 mS), delay (0.02 mS) and frequency (20 pps). The

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last part of the experiment was to find out the effects of epinephrine by observing and
recording heart activities, after the heart had completely recovered from vagal stimulation for
5 minutes.

RESULTS
During the first part of the experiment, after the insertion of wire into the ventricle, the
frog heart stopped beating for around one minute. With firm massages of the liver and the
heart, the heart beat recovered.
The mechanical signal and the electrical activity were recorded for a total of two
minutes. For the first 85 seconds, signals of atrial contraction and ventricular contraction
were observed clearly, and the force of ventricular contraction was largely greater than atrial
contraction (Figure 1). During the first 85 seconds, the average force of atrial contraction was
10.14 grams, the average force of ventricular contraction was 11.25 grams, and the heart rate
was 35 BPM.

The first 85 seconds of heart activity.

Force (grams)
Time (seconds)

Figure 1. The first 85 seconds of heart activity. The average force of atrial contraction was

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10.14 grams, the average force of ventricular contraction was 11.25 grams, and the heart rate
was 35 BPM.
During the period from 85 seconds to the end of record, there was a sudden rise in
contraction force for 10 seconds, and atrial contractions were not clearly observed (shown in
Figure 2). After 95 seconds, contractions became stable, the average force of atrial
contraction was 10.15 grams, the average force of ventricular contraction was 11.30 grams,
and the heart rate was 38 BPM.

Heart activity between 85 seconds and 120 seconds.

Force (grams)
Time (seconds)

Figure 2. Heart activity between 85 seconds and 120 seconds. For the first 10 seconds,
contraction forces increased, and the overall rate and rhyme were irregular. Atrial
contractions were not observed at that time. After 95 seconds, the overall rate became stable,
the average force of atrial contraction was 10.15 grams, the average force of ventricular
contraction was 11.30 grams, and the heart rate was 38 BPM.

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Latency was the time between the initiation of electrical activity and the beginning of
ventricular contraction. However, the electrical activity of the whole process was not clearly
formed, so latency could not be determined at this time.
In order to elicit extrasystolic contraction, the frog heart needed to be stimulated by
gradually increased stimulus voltage until the threshold voltage was reached. The threshold
voltage was the minimal voltage that could elicit an extrasystolic contraction during late
diastole. The maximum stimulus voltage was 20 volts; however, extrasystolic contraction was
not elicited even with the maximum stimulus voltage. Extrasystolic contraction did not
present as well during two times the threshold during late diastole, which was set at 40 volts.
During early diastole, no extrasystolic contraction was elicited with increasing stimulus
voltage (the maximum stimulus voltage was set at 50 volts). And no compensatory pause was
observed when no extrasystolic contraction was elicited.
In the next part of the experiment, the vagus nerve was stimulated directly by the
electrode in order to elicit bradycardia. The stimulus voltage that elicited bradycardia was
0.63 volts. Table 1 compares the heart rate and contractile force before and after vagal
stimulation during bradycardia.
According to Table 1, the heart rate before vagal stimulation during bradycardia was 36
BPM, which was twice as the heart rate after vagal stimulation. The heart rate decreased 50%
following vagal stimulation. The contractile force before vagal stimulation during
bradycardia was 0.40 grams, which was almost twice as the contractile force after vagal
stimulation (0.21 grams).

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Table 1. Comparison of heart rate and contractile force before and after vagal stimulation
during bradycardia.
Heart Rate (BPM)

Contractile force (grams)

Before vagal stimulation during bradycardia

36

0.40

After vagal stimulation during bradycardia

18

0.21

Table 1. The heart rate and contractile force were collected and measured before and after
vagal stimulation during bradycardia by BioPac. BPM stands for beats per minute. The heart
rate decreased 50% following vagal stimulation.
Then the vagus nerve was stimulated with gradually increased stimulus voltage in order
to elicit cardiac arrest. The stimulus voltage at the point of heart arrest was 0.69 volts. Table 2
shows the heart rate and contractile amplitude before and after vagal stimulation.
Table 2. Comparison of the heart rate and contractile amplitude before and after vagal
stimulation.
Heart Rate (BPM)

Contractile amplitude (grams)

Before vagal stimulation

39

0.09

After vagal stimulation

34

0.08

Duration of cardiac arrest

43.19 seconds

Table 2. Heart rate and contractile force were collected and measured by BioPac. BPM
stands for beats per minute. The heart rate decreased 12.82% following vagal stimulation.
According to Table 2, the heart rate before vagal stimulation was 39 BPM, and the

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heart rate after vagal stimulation was 34 BPM. The heart rate decreased 12.82% following
vagal stimulation. The contractile amplitude before vagal stimulation was 0.09 grams, which
was larger than the contractile amplitude after vagal stimulation (0.08 grams). Cardiac arrest
lasted for 43.19 seconds.
In the last part of the experiment, after the heart recovered for 5 minutes from vagal
stimulation, in order to find out the effects of epinephrine, 25 drops of epinephrine were
added to the ventricle. Table 3 shows the heart rate and contractile amplitude before and after
adding epinephrine.
Table 3. Comparison of the heart rate and contractile amplitude before and after adding
epinephrine.
Heart Rate (BPM)

Contractile amplitude (grams)

Before adding epinephrine

39

0.88

After adding epinephrine

30

2.80

Table 3. Heart rate and contractile amplitude were collected and measured before and after
adding epinephrine by BioPac. BPM stands for beats per minute. The heart rate decreased
23.08% after the effects of epinephrine were observed.
According to Table 3, the heart rate before adding epinephrine was 39 BPM, which was
higher than the heart rate after adding epinephrine (30 BPM). The heart rate decreased
23.08% after the effects of epinephrine were observed. The contractile amplitude after adding
epinephrine was 2.80 grams, which was much greater than the contractile amplitude before
adding epinephrine (0.88 grams). According to Figure 3, the trend of contraction force after

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applying epinephrine to the ventricle rapidly increased at the first 65 seconds, then reached a
plateau for about 50 seconds, and then decreased gradually.

Figure 3. The overall trend of contractions after applying epinephrine to the ventricle. The
contraction force increased rapidly at first, then reached a plateau, and gradually decreased.
Data was collected for 4 minutes after adding epinephrine.

DISCUSSION
In the first part of the experiment, it was expected to be observed that ventricular
contractions were larger than atrial contractions, and the results were consistent with the
expectations. The results were also consistent with the other research that demonstrated
ventricle contraction generated larger action potential than atrial contraction (Jaakko et al.,
2014).The reason that force of ventricular contraction was larger was because the ventricle
needed to pump blood to the lungs or the rest of the organs of the body. The ventricle needs
larger force than atria, which pump blood to the ventricles. Also, because the ventricle is

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larger than the atrium, it has larger contraction forces. The sudden increase in contraction
force at 85 seconds may be because someone touched the table while recording data, changed
the baseline tension, and elicited irregular rate.
No clear and regular electrical activities were observed during the whole process of the
experiment. This may be because during the process of preparation, the spring clip was set to
far away from the heart.
In the next part of the experiment, no extrasystolic contraction was generated during
late diastole stimulation because of the following reasons. First, the frog heart stopped
beating for one minute at the beginning of the experiment; although the heart recovered after
firm massages, the heart was still very weak throughout the whole experiment. Second, there
was not enough filling time for the ventricles to generate another contraction because the
venous return to the heart was not enough. According to Frank Starling Law of the Heart, low
venous return results in low stroke volume, so there was not enough action potential to
generate another contraction. Third, the stimulation was applied too early, before the period
of late diastole. Theoretically, stimulations during late diastole could elicit extrasystolic
contractions. Extrasystolic contractions are premature ventricular contractions, and during
late diastole, the heart is in the relative refractory period. With a large enough stimulus,
another action potential could be generated during the relative refractory period. However, no
extrasystolic contraction should be present during early diastole stimulation, because at that
time, the heart is in the absolute refractory period, which means no matter how large the
stimulus, no action potential could be generated. Extrasystolic contraction force should be
smaller than regular ventricular contraction because there is less filling time. According to

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Frank Starling Law of the Heart, the greater filling of the ventricle, which is more venous
return to the heart, leads to increased contractility and results in greater contraction during
systole (Holly A. Shiels and Ed White, 2005). Theoretically, the force of extrasystolic
contraction would not change no matter how large the stimulus is. So even with two times the
threshold voltage during late diastole, the extrasystolic contraction force would be the same
as the condition with threshold voltage. Previous research demonstrated that extrasystolic
contraction was possible to elicit during late diastole (Nakao et al., 1994). After the
extrasystolic contraction, there would be a compensatory pause until the next ventricular
contraction. The compensatory pause is because the extrasystolic contraction leads to its own
refractory period, so there are prolonged filling times. The ventricular contraction which
follows extrasystolic contraction is usually larger than normal contraction because of the
Frank Starling Law of the Heart. After the compensatory pause, stroke volume increases, so
the force of the contraction increases as well.
In the next part of the experiment, the heart rate decreased from 36 BPM to 18 BPM,
which was 50% off, after the direct vagal stimulation during bradycardia, and the contractile
force decreased from 0.40 grams to 0.21 grams as well. This result was consistent with
previous research that demonstrated direct vagal stimulation would stop pacemaker potentials
and decrease the heart rate (A R Bywater et al., 1990). The vagus nerve is part of the
parasympathetic nervous system; with direct stimulation (0.63 volts) of the vagus nerve, the
parasympathetic nervous system would release acetylcholine (Ach) to bind to the muscarinic
receptor and cause bradycardia (Suhayla et al., 2001). The parasympathetic nervous system
also increases the permeability of potassium ions in the SA node in human hearts or sinus

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venosus in frog hearts, to slow the pacemaker. The decrease in contractile force was caused
by the inhibition of L-typed Ca2+ channels, which would reduce Ca2+ influx and result in
weakened atrial contraction (Sherwood, 2010, p. 326).
By increasing the stimulus voltage to 0.69 volts, cardiac arrest occurred. The total
duration of cardiac arrest was 43.19 seconds. The stoppage of the heart was because the main
pacemaker on the sinus venosus was impacted by over stimulation. A period after the heart
stopped beating, the heart gradually resumed at a lower rate under continuous vagal
stimulation. Vagal escape occurred. During vagal escape, other pacemaker cells took over
control to initiate the rhythm of the heart while the main pacemaker cells on the sinus
venosus functioned improperly. The reason that the heart rate after vagal stimulation (34
BPM) was slower than before (39 BPM) and the contractile force decreased was because the
other pacemakers were not as fast as the main pacemakers on the sinus venosus. This result
was consistent with previous research, which demonstrated that other pacemaker cells such as
pacemaker cells on AV node in the dogs heart would take over control of the heart rate
during vagal escape (Andrew G. Wallace and Willard M. Daggett, 1964).
In the last part of the experiment, epinephrine was applied to the ventricle. Epinephrine
is released by the sympathetic nervous system, by binding to beta-adrenergic receptors.
Epinephrine would enhance the heart contractility and increase the strength of the ventricular
contraction (Sherwood, 2010, p. 328). According to Table 3, the contractile amplitude
increased largely from 0.88 grams to 2.80 grams with the presence of epinephrine; however,
the heart rate decreased from 39 BPM to 30 BPM. The heart rate decreased because the
increase in contractile amplitude was too rapid and large. However, theoretically, both of the

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heart rate and contractile amplitude should increase. According to Figure 3, after the rapid
increase in the contraction force within 50 seconds, the contraction force stayed constant for
around 30 seconds and then gradually decreased. This might be because of the decrease in the
effects of epinephrine.
Norepinephrine, which has some commonalities with epinephrine, would increase Ca2+
influx into the cytosol. Greater cytosolic Ca2+ would lead to greater heart contraction force
(Sherwood, 2010, p. 329). So, both the norepinephrine and epinephrine would increase the
force of contraction, increase pacemaker, and cause tachycardia. The effects of epinephrine
were proven in the previous research which demonstrated that epinephrine would increase the
heart rate, also have significant functions during the period of cardiac arrest and could be
used for CPR ( Mark et al., 2008).

CONCLUSION
Electrical and mechanical properties of the frog heart were investigated during this
experiment. The force of ventricular contraction was generally larger than the force of atrial
contraction. Stimulus during late diastole could elicit extrasystolic contraction while stimulus
during early diastole could not. The extrasystolic contraction force would not be changed by
increasing stimulus voltage. The parasympathetic nervous system acts via the vagus nerve,
and releases Ach to decrease the heart rate and contractile force. The sympathetic nervous
system acts via norepinephrine, also releases epinephrine, and both of these hormones would
increase heart rate and contractile force.

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REFERENCE
1. Sherwood, Lauralee. Human Physiology: From Cells to Systems. 2010. 7th ed.
2. Subramani Sathya, Mammen Priya. Letter to the Editor, Extra Systoles in the Frog Heart.
2001. Indian J Physiol Pharmacol 2001; 45 (4) : 511-513
3. Berntson Gary, Quigley Karen, Fabro Vincent and Cacioppo John. Vagal stimulation and
cardiac chronotropy in rats. 1992. Journal of the Autonomic Nervous System, 41(1992)
221-226
4. Hsieh JH, Pan CM, Kuo JS, Chai CY. Predominance of vagal bradycardia mechanism in
the brain stem of turtles. J. exp. Biol. 140, 405-420 (1988)
5. Bautista Erwin, Korber Julia. NPB 101L Physiology Lab Manual. 2009
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Basal Vertebrate, the European River Lamprey (Lampetra fluviatilis). Physiological and
Biochemical Zoology, Vol. 87, No. 6 (November/December 2014), pp. 817-828
7. Shiels A Holly, White Ed. The FrankStarling mechanism in vertebrate cardiac myocytes.
The Journal of Experimental Biology 211, 2005-2013. doi:10.1242/jeb.003145
8. S. NAKAO,T. TOMARIJ, and H. TANAKA. Influence of left ventricular filling profile
during preceding control beats on pulse pressure during ventricular premature contractions.
European heart journal 15 (1994): 462-467.
9. Bywater R A, Campbell G D, Edwards F R, et al. Effects of vagal stimulation and applied
acetylcholine on the arrested sinus venosus of the toad[J]. The Journal of physiology, 1990,
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10. Mukaddam-Daher S, Yin Y L, Roy J, et al. Negative inotropic and chronotropic effects of

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oxytocin[J]. Hypertension, 2001, 38(2): 292-296.

11. WALLACE A G, DAGGETT W M. Pacemaker activity during vagal escape rhythms[J].
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Raw Data
Heart activity

Threshold during late diastole

19

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2 threshold during late diastole

Threshold during early diastole

20

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Bradycardia

Vagal Escape

21

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Epinephrine

22