Alexandra Hebert

BIOL 4161
Section 002
25 November 2019

The effects of myogenic cardiac activity, the Frank-Starling mechanism, autonomic

control, temperature, and taurine on the heart of the African claw frog, Xenopus laevis.

Abstract

The amphibian heart contains three chambers rather than the four in humans. Using the heart of

the Xenopus laevis, we used myogenic cardiac activity, the Frank-Starling mechanism,

autonomic control, temperature, and taurine’s effects on the contraction amplitude and heart rate

of the cardiac muscle as a means of gaining a deeper understanding of cardiac physiology. A

latency period was examined to be high enough for proper AV nodal delay. Imposing a stretch

on the cardiac muscle utilized the Frank-Starling mechanism and resulted in an increased cardiac

contractile amplitude with increased stretch. Autonomic control caused an increase in contractile

amplitude and heart rate upon sympathetic input, but the parasympathetic input only decreased

heart rate and atrial contraction amplitude while the ventricular contractile amplitude increased

when acetylcholine was added. Three different Ringer’s solution were used to test temperature’s

effect on cardiac activity, and it was found increasing temperature of the solution increased both

contractile amplitude and heart rate. These results were cohesive with previous experiments.

Addition of taurine at a 15 mM concentration caused a decrease in contractile amplitude of the

ventricle while the 30 mM concentrated taurine caused an increase in the contractile amplitude.

Taurine had no effect on heart rate and has homeostatic qualities.

 Introduction

While the amphibian heart has only three chambers rather than the four present in mammalian

hearts, the two atria and one ventricle comprising the pumps work together to ensure proper blood

flow. Pacemaker cells of the amphibian heart are known as sinus venous, and deoxygenated blood

empties into the right atrium through this pacemaker cell. Oxygenated blood is emptied into the

left atrium through the pulmonary vein, while the one ventricle receives blood from both atria and

pumps it out through the conus arteriosus. There is little mixing of the blood due to the structural

organization of the ventricle and conus arteriosus. We used myogenic cardiac activity, the Frank-

Starling mechanism, autonomic control, temperature, and taurine’s effects on the contractile

activity and heart rate of the cardiac muscle from the African claw frog, Xenopus laevis, in order

to gain a deeper understanding of cardiac muscle physiology.

Wholehearted contractions are coordinated by the spread of electrical conduction beginning with

the depolarization of the sinoatrial node that then spreads via the internodal pathway. The

atrioventricular node delays the signal and allows enough time for the depolarization to spread

through atria via gap junctions resulting in contraction of both atria before contraction of the

ventricles. Latency period is the time between the atria contraction and ventricular contraction.

The depolarization then spreads rapidly through the Bundles of His and Purkinje fibers and

continues travelling upward through the ventricles and causes them to contract. Using surface

electrodes to record the electrical activity of the heart helped determine the spread of activity that

was summed and expressed as results from an electrocardiogram (ECG). An ECG was used to

determine the baseline levels of cardiac activity in addition to investigating the temporal

relationship between the mechanical and electrical events that occur during each cardiac cycle.
The Frank-Starling mechanism is an intrinsic mechanism to prevent the accumulation of blood in

the heart. Due to an increased end-diastolic volume (EDV) stretching the myocardial cells of the

ventricular wall, an increase in EDV causes a proportional increase in stroke volume (SV). The

length of a resting sarcomere determines the magnitude of the contractile force at a given EDV,

which is positively correlated with the corresponding SV. By increasing overlap of thick and thin

filaments, a subsequent increase in the contractile force also results. In order to determine the

ventricular contractile amplitude of the cardiac muscle, the Frank-Starling mechanism was

explored by manually stretching the heart.

Input from the autonomic nervous system causes significant changes to cardiac muscle activity.

Addition of acetylcholine (Ach) to cardiac muscle will decrease its activity because the binding

of Ach to muscarinic receptors decreases the ion probability of T-type calcium and funny sodium

channels, while increasing the ion flux of potassium leak channels to cause a decrease in

membrane potential. Decreasing the membrane potential of the cell will result in a decrease in

contractile activity. Atropine and epinephrine are chemicals that activate the sympathetic nervous

system by binding to adrenergic receptors on the pacemaker and contractile cells of cardiac

muscle and initiates the cAMP pathway to activate PKA, which triggers the opening of T-type

calcium and funny sodium channels to create a positive ion flux to increase membrane potential

of the cell. This influx will increase the drift to threshold of the action potential and thus cause an

increase in heart rate. Through addition of epinephrine, acetylcholine, and atropine, the effects of

the autonomic nervous system on cardiac muscle was explored.

We investigated both temperature and taurine’s effect on ventricular contractile activity and heart

rate. Our hypothesis was that a temperature increase will result in an increase in both contractile

strength and heart rate, while the same will result with increasing taurine concentrations. The

opposite will occur when temperature is decreased.

Methods

Experiments investigating myogenic cardiac activity, the Frank-Starling mechanism, an

autonomic control of the heart were performed in accordance with the methods outlined

(Boswell Fall 2019).

Another experiment was performed to explore the effects of temperature and taurine on

ventricular contractile strength and heart rate of the cardiac muscle of the X. laevis. Equipment

set up and calibration of the force transducer were performed as outlined in (Boswell Fall 2019).

The positive, negative, and ground leads were attached to the appropriate locations on the left

forelimb, right forelimb, and right hindlimb dissecting pins. A fishhook was inserted through the

apex of the ventricle of the heart muscle and connected to the force transducer using a string.

Click on the units drop down menu and select “g”. Cardiac muscle was moistened with cold (4

°Celsius) Ringer’s solution to cool down the muscle. Rinse every ten seconds with cold Ringer’s

for ninety seconds. Wait one minute. Moisten cardiac muscle with room temperature (25

°Celsius) Ringer’s solution every ten seconds for ninety seconds. Wait one minute. Moisten

cardiac muscle with hot (35 °Celsius) Ringer’s solution every ten seconds for ninety seconds.
 Results

Myogenic cardiac activity: Upon measurement of the electrical and mechanical events found in

each cardiac cycle of the X. laevis, the latency between the QRS complex and ventricular

contraction is 493 ± 150 milliseconds (ms).

Frank-Starling mechanism: Imposing a stretch on the heart of the African claw frog caused a

corresponding increase in the ventricular contraction amplitude with the 1 mm-imposed stretch

bar representing no stretch at all and 11 mm serving as an imposed stretch of 10 mm. The first

ventricular contraction amplitude measured was 0.326 ± 0.0013 g as depicted in the first bar of

the graph (Fig. 1). Upon stretching 10 mm, as seen in bar 11, the ventricular contraction

amplitude was 1.41 ± 0.01 g (Fig.1). A direct relationship was identified where the magnitude of

the ventricular contraction amplitude increased as the imposed stretch on the heart increased

(Fig. 1).
 1.6

Ventricular Contraction Amplitude (g)

1.4

1.2

0.8

0.6

0.4

0.2

0
1 2 3 4 5 6 7 8 9 10 11
Imposed Stretch (mm)

Figure 1: The ventricular contraction amplitude (g) of the heart of the

Xenopus laevis resulting from an imposed stretch (mean ± SD, n = 3).
Contractile amplitudes of the ventricles are displayed on the Y-axis
and were measured in grams (g). Imposed stretch is expressed on the
X-axis and was measured in millimeters (mm). Stretch on the cardiac
muscle: 1 mm serves as the position of no imposed stretch and 11 mm
represents a 10 mm.

Autonomic control-atrial contraction: Adding chemicals that affect the autonomic nervous

system to the cardiac muscle of the X. laevis resulted in varying levels of activity with respect to

each chemical. The addition of epinephrine and atropine resulted in atrial contraction amplitudes

of 0.153 ± 0.033 g and 0.151 ± 0.04 g respectively (Fig. 2). Acetylcholine addition caused an

atrial contraction amplitude of 0.136 ± 0.021 g. While acetylcholine decreased the atrial

contraction amplitude, epinephrine and atropine increased the contractile activity of the atria

(Fig. 2).
 0.25

Atrial Contraction Amplitude (g)

0.2

0.15

0.1

0.05

0
Epinephrine Acetylcholine Atropine
Added Chemicals

Figure 2: The atrial contraction amplitude (g) of the cardiac muscle of

the X. laevis due to added chemicals (mean ± SD, n = 3). Atrial
contraction amplitude is expressed on the Y-axis and was measure in
grams (g). The X-axis displays epinephrine, acetylcholine, and
atropine, which were chemical added to the heart muscle.

Autonomic control-ventricular contraction: The addition of epinephrine, acetylcholine, and

atropine resulted in varying effects on the ventricular contraction amplitudes of the X. laevis

heart. The control contractile amplitude of the ventricles was 0.379 ± 0.0175 g (Fig. 3). Upon

addition of the three chemicals, the epinephrine resulted in an amplitude of 0.747 ± 0.126 g,

acetylcholine caused an amplitude of 0.795 ± 0.031g, and atropine produced an amplitude of

0.822 ± 0.0649 g. The results depict an increase in contractile amplitude of the ventricles upon

addition of all three chemicals with respect to the control (Fig. 3).
 1

ventricular Contraction Amplitude (g)

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control Epinephrine Acetylcholine Atropine
Added Chemicals

Figure 3: Ventricular contraction amplitude (g) of the heart muscle of

the X. laevis due to the addition of chemicals (mean ± SD, n=3). The
varying levels of ventricular contraction amplitude are displayed on
the Y-axis and measured in grams (g). Added chemicals are expressed
on the X-axis. Control represents the ventricular contractile amplitude
produced in the absence of additional chemicals.

Autonomic control-heart rate: Upon addition of chemicals that affect the autonomic nervous

system of the X. laevis, the heart varied from the control. With the control heart rate being 30 ± 1

BPM, the addition of epinephrine to the cardiac muscle cause an increase in heart rate at 34 ± 1

BPM (Fig. 4). Acetylcholine added after the epinephrine caused a decrease in heart rate with

22.7 ± 2.08 BPM. After acetylcholine, atropine was added and increased the heart rate to 38.3 ±

0.58 BPM. Epinephrine and atropine addition caused a significant increase in the heart rate,

while acetylcholine decreased the heart rate in relation to the control (Fig. 4).
 45
40
35

Heart Rate (BPM)

30
25
20
15
10
5
0
Control Epinephrine Acetylcholine Atropine
Added Chemicals

Figure 4: Heart rate (BPM) of the cardiac muscle of the X. laevis in

the presence and absence of additional chemicals (mean ± SD, n=3).
Added chemicals and the control are expressed on the X-axis. Heart
rate is displayed on the Y-axis and is measured in beats per minute
(BPM). Control represents the heart rate in the absence of added
chemicals.

Temperature-ventricular contraction: Temperature’s effect on ventricular contraction amplitude

of the heart from the X. laevis was measured using three different temperatures of Ringer’s

solution. Washing the heart with 4 °C Ringer’s solution produced a ventricular contractile

amplitude of 0.151 ± 0.016 g (Fig. 5). Adding 25 °C and 38 °C Ringer’s solution to the heart

increased the contractile amplitude to 0.349 ± 0.033 g and 0.358 ± 0.033 g respectively.

Increasing temperature of the Ringer’s solution increased the contractile amplitude of the

ventricles (Fig. 5).

 0.45
0.4
Ventricular Contraction Amplitude (g)
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
4° 25° 38°

Temperature of RInger's Solution (°C)

Figure 5: Ventricular contraction amplitude (g) of the cardiac

muscle of the X. laevis due to varying temperatures of Ringer’s
solution (mean ± SD, n = 3). The Y-axis depicts the contraction
amplitude of the ventricles in grams (g). Temperatures of 4, 25, and
38 degrees Celsius are expressed on the X-axis.

Temperature-heart rate: Washing the heart of the X. laevis with different temperature Ringer’s

solution was used to determine temperature’s effect on heart rate. The addition of 4 °C Ringer’s

solution produced a heart rate of 32 BPM (Fig. 6). When the heart was washed with 25 °C

Ringer’s solution, the heart rate increased to 40 BPM. As a 38 °C Ringer’s solution was applied

to the heart, the heart rate further increased to 48 BPM. With increasing temperature of Ringer’s

solution, a corresponding increase in heart rate occurred (Fig. 6).

 60

50
Heart Rate (BPM)
40

30

20

10

0
4° 25° 38°
Temperature of Ringer's Solution (°C)

Figure 6: Heart rate of the X. laevis due to varying temperatures of

Ringer’s solution (n=1). The Y-axis expresses heart rate in beats per
minute (BPM). X-axis displays temperatures of Ringer’s solution at
4, 25, and 38 degrees Celsius.

Taurine addition-ventricular contraction: Adding different concentrations of taurine to the

cardiac muscle of the African claw frog was used to investigate its effects on the ventricular

contraction amplitude in relation to a control. With the control amplitude at 0.33 ± 0.016 g, the

addition of 15 mM taurine decreased the amplitude to 0.273 ± 0.016 g (Fig. 7). The addition of

30 mM taurine increased the contractile amplitude of the ventricles to 0.321 ± 0.016 g. The

lower concentration of taurine caused a decrease in amplitude, while the higher concentration

increases the amplitude (Fig. 7).

 0.4

0.35

Ventricular Contraction Amplitude (g)

0.3

0.25

0.2

0.15

0.1

0.05

0
Control 15 mM Taurine 30 mM Taurine
Added Chemicals

Figure 7: Ventricular contraction amplitude (g) of the cardiac muscle

of the X. laevis in response to differing concentrations of taurine. Y-
axis expresses the contractile amplitude of the ventricles in grams (g).
X-axis expresses the added chemicals measured in milli-molars (mM).
Control represents the ventricular contraction amplitude in the absence
of taurine.

Taurine-heart rate: Different concentrations of taurine added to the cardiac muscle of X. laevis

was used to observe its effects on heart rate with respect to the control. After adding 15 mM and

30 mM taurine to the heart, no variation was found to occur from the control heart rate at 36

BPM (Fig. 8).

 40

35

30
Heart Rate (BPM)
25

20

15

10

0
Control 15 mM Taurine 30 mM Taurine
Added Chemicals

Figure 8: Heart rate of the X. laevis due to increasing concentrations

of taurine (n=1). The Y-axis displays the heart rate in beats per minute
(BPM). Added chemicals are expressed on the X-axis. Control
represents the heart rate measured in the absence of taurine.

Discussion
Myogenic cardiac activity: The temporal relationship between the measured electrical and

mechanical cardiac activity was examined as the latency between the QRS complex and

ventricular contraction at 493 ± 150 ms. This period of latency allows for the AV nodal delay so

both atria contract before the ventricles. One limitation we encountered was the inability to

obtain an atrial contractile amplitude control.

Frank-Starling mechanism: The cardiac muscle was found to express a direct relationship

between imposed stretch and ventricular contraction amplitude (Fig. 1). Stretching the muscle

stimulates an increased EDV where the measured contractile force serves as an indicator of SV.

This positive relationship between the two variables agree with our basic understanding of the
principles behind the Frank-Starling mechanism. Future experiments that utilize the Frank-

Starling mechanism is different types of muscles could be useful to explore.

Autonomic control of the heart: Heart rate, ventricular contractile activity, and atrial contractile

activity all increased with the addition of epinephrine and atropine (Fig. 2,3,4). This direct

relationship agrees with our current knowledge of the sympathetic nervous system’s effect on

cardiac activity where an increase in sympathetic input will result in an increase in cardiac

activity. We found that the heart rate and atrial contraction rate decreased upon addition of

acetylcholine, the parasympathetic input (Fig. 2,4). These results match our present knowledge

of the effects parasympathetic input on cardiac physiology. However, input of acetylcholine

increased the ventricular contractile amplitude, and this does not agree with our current

knowledge of parasympathetic input on cardiac physiology. Since it was tested immediately

before, the epinephrine may have left a residual excitation response causing the acetylcholine to

have an opposite effect than intended. Strictly focusing on autonomic effects on pressure could

be tested in future experiments.

Temperature effect on cardiac activity: We found the cardiac muscle of the X. laevis to exhibit

temperature sensitivity to a variety of temperatures resulting in an increase in contractile

amplitude with an increase in temperature, which supports our hypothesis (Fig. 5,6). These

results agree with previous studies performed on the cardiac muscle of Homo sapiens which

report an increase in heart rate upon increase in heat stress (Wilson and Crandall 2011).
Taurine effect on cardiac activity: Upon addition of 15 mM taurine to the cardiac muscle of X.

laevis, the ventricular contractile amplitude decreased, while the higher taurine concentration at

30 mM increased the contraction magnitude. The margin of error for the control and both

concentrations of taurine were all the same (Fig. 7,8). Taurine had no effect on heart rate.

Previous experiments agree report that taurine possesses homeostatic functions and out results

agree (Schaffer et al. 2010). Our hypothesis that taurine would increase the heart rate and

contractile amplitude was incorrect. Future experiments could be to investigate taurine’s effect

on heart rate in the presence of caffeine.

The heart of the African claw frog serves as an ideal organ for investigation into cardiac

physiology of vertebrates. We determined that cardiac muscle is sensitive to temperature at the

levels of contractile amplitude and heart rate. Taurine was found to have homeostatic functions

through its maintenance of heart rate and contractile amplitude at the higher concentration of 30

mM. These experiments allowed for direct observation of cardiac physiology through myogenic

cardiac activity, Frank-Starling mechanisms, autonomic control, temperature, and taurine’s effect

on cardiac muscle, which expanded our understanding of cardiac physiology.

 Literature Cited
Boswell LC (Fall 2019) Vertebrate Physiology Student Lab Manual. Baton Rouge, LA:

Louisiana State University.

Wilson TE, Crandall CG (2011) Effect of Thermal Stress on Cardiac Function. Exercise and

Sports Sciences Reviews 39 (1): 12–17.

Schaffer SW, Jong CJ, Ramila KC, and Azuma J (2010) Physiological Roles of Taurine in Heart

and Muscle. Journal of Biomedical Science, 17(Suppl 1): S2.