Rana pipiens Heart Rate and Force Contractile Under Various Treatment: Temperature, Stretch,

Dugs, and Conduction Blockade

Name: HsiuTing Chiu

Student Number: 213583935
BIOL 3060 Lab 05
TA: Leah Turner
 Due Date: Nov 9, 2016
Abstract

Understanding heart rate and contractile force of the cardiac muscle is very important as they can

show us how the heart regulates and accommodate itself under different conditions such as

temperature, muscle stretch, drug influence, and conductor blockade. Using Rana pipiens heart

for this experiment, we hypothesized that the heart rate will increase when the salin solution that

bathed the heart is around 40°C and decrease when the solution is at 4°C. Also, we hypothesized

that the heart rate will decrease and contractile force will increase as the heart is under a higher

degree of stretch. The addition of acetylcholine and pilocarpine were hypothesized to decrease

the heart rate. In contrast, the addition of epinephrine and atropine increase the heart rate. Lastly,

the hypothesis stated that a greater degree of blockade increase the time interval between atrial

conduction and ventricular conduction. The experiment was carried out in situ with the force

transmitter connected to the heart of the frog by pins. The heart rate and contractile force was

measured by using Lab chart showing the electrocardiogram and force channel. The results

shown that the increase in temperature did indeed increase the heart rate and lowering the

temperature decreased the heart rate. It also showed that the increase in stretch, increase the

contractile force and decrease the heart rate. Measuring the heart rate showed that it increased

when epinephrine and atropine was added to the heart. On the other hand, the addition of

acetylcholine and pilocarpine decreased the heart rate. Finally, the blockade on the

atrioventricular node of the atria increased the time interval between the atrial conduction and

ventricular conduction. By learning about the heart rate and contractile force under different

conditions of a frog’s heart, we can apply the knowledge onto humans and understand how to

treat some heart problem such as the heart block that some people suffer.
Introduction

The muscular heart is essential for living. The pumping of the heart regulates the blood

flow to circulate through all parts of the body to distribute oxygen (Charles 1977). It is made up

of one of the three kinds of muscles: cardiac muscles and the heart is controlled by the

autonomic nervous system (Cannell et al. 1995). Cardiac muscles are similar to the skeletal

muscle in terms of the features and contraction mechanisms but different in the unique feature of

rhythmicity, which is made possible by gap junctions (Joseph et al. 1907). The cardiac muscle

cells, cardiomyocytes, allows the electrical current flow in cardiac muscle from cell to cell

through the gap junction which creates a rhythmic pattern therefore give rise to the heartbeats

(James 1991). It is because of the gap junction and the close relation of the cells, the entire heart

can be elicited by stimulation on a single cardiomyocyte (Norbert et al. 2003).

The heart can produce myogenic contraction, which means that the myocyte cells can

initiate contraction itself without the outside stimulus (McCarron et al. 1997). This is possible

due to the leak of sodium (Na+) and calcium ions (Ca2+) into the cell (Xun et al. 2005). This is

enough to reach the action potential threshold to cause depolarization. This cause the activation

of ryanodine receptors to induce the release of Ca2+ from the sarcoplasmic reticulum (Cannell et

al. 1995). Calcium ions then bind to the troponin causing the conformation change on actin to

expose the myosin binding site (Fuchs et al. 1970). Myosin head binds onto actin filament and

produce the power stroke. The power stroke causes the shortening of the muscle and therefore

contract (Gordon et al. 2001). The cells are then repolarized by the influx of potassium ions (K+)

through voltage-gated channels (Harvey et al. 1970).

The Rana pipiens heart was used for this study. It has three chambers unlike the humans,

which have four. The three chambers include two artia and one ventricle (Deepak et al. 2000).
The flow of action potential allows the atrial conduction to occur before the ventricular

conduction (Thomas et al. 1963). This produce the PR interval, which the conduction of atrial

can be seen earlier than ventricular on the graph conducted by measuring the heart beat with

electrocardiograms (ECG). The mid line that separates the ventricle and artia is the

atrioventricular (AV) node.

The objective of this experiment was to look at how the heart adjust and behave when

subjected to different conditions. The heart rate and contractile were measured under various

temperature, stretch, drug, and blockade. The hypothesis stated that the heart rate of r. pipiens

will increase and decrease accordingly to the surrounding’s temperature. The higher the

temperature the faster the heart beats and the lower the temperature, the slower the heart beats. It

was also hypothesized that the force of contractile will increase as the muscular stretch

increased. On the other hand, the heart rate will decrease when the stretch increased. Another

hypothesis stated that the heart rate decreased with the addition of acetylcholine or pilocarpine

and increased with the addition of epinephrine or atropine. The last hypothesis stated that as the

degree of conduction blockade increased, the time interval between the atrial and ventricular

conduction increased to a point where there is no signal transmission.

Materials and Methods

Lab 6 of the lab manual, Frog Heart, was used to perform this experiment and nothing was

changed in the protocol (AD Instruments 2010).

Results

The contractile force and heart rate of Rana pipiens heart rate in situ was observed under

different conditions such as stretching of ventricular muscle, various drugs treatment, blockade,

and different temperature. These were compared to the control baseline heart rate.

The baseline heart rate was experimented while the heart was at resting condition. Figure

1 depicted the heart rate in beats per minute (BPM), all calculated from the force channel. The

calculated mean heart rate of was 68.05BPM. Also, the value of maximum heart rate was at

288.428BPM and the minimum was 17.44BPM.

Heart rate was observed when Ringer’s solution of different temperature was used on the

heart. The effect was shown in Figure 2. Mean heart rate was calculated and graphed. The

normal room temperature at 21°C is the control in this experiment and it gave a heartbeat rate of

64.157BPM. The warm Ringer’s solution was at 38.5°C and it rose to 81.49BPM. The cold salin

solution was at 4°C and the heartbeat rate dropped to 46.50BPM.

Tension was exerted onto the heart muscle by stretching it. It was stretched by an

increment of 0.5mm every time for a total of 5 times. Figure 3 shows the amount of force related

to the stretch. The scatter plot shows an increasing trend by looking at the line of best fit. The

correlation coefficient also shows the strong positive correlation trend. The highest stretch at

42.5mm shows the highest contractile force at 0.0094N. Addition to the contractile force, the

mean heart rate was also measured at different stretch tension. This data can be seen in Figure 4.
The mean of the heart rate shows a decreasing trend and increase when it reaches 57BPM. The

overall trendline is decreasing as seen in the figure. The coefficient shows a weak negative

correlation.

Effects of different drugs on heart rate was measured and was shown in figure 5. The

percent change in heart rate was shown and the values were calculated by using the change

between before and after the addition of drug (calculation in Appendix). Acetylcholine has a

value of -28.3% which is the smallest among all the drugs. Epinephrine however, has a value of -

7.24% made it the biggest change in heart rate among all the drugs.

The time intervals between the atrial contraction and ventricular contraction of the heart

was measured and calculated in Figure 6. The longest time taken was in third-degree block at

0.51s and the shortest was at first degree block at 0.38s. Standard error of mean are shown by

error bars and they do not overlap each other.

 350

300

250
Heart Rate (BPM)

200

150

100

50

0
Mean Heart Rate (BPM) Maximum Heart Rate (BPM) Minimum Heart Rate (BPM)

Figure 1. Comparison of Resting Heart Rate. The bar graph shows the mean, maximum and
minimum heart rate of Rana pipiens in a resting state.

90

80

70
Mean Heart Rate (BPM)

60

50

40

30

20

10

0
Normal Salin (21°C) Warm Salin (38.5°C) Cold Salin (4°C)
Salin Temperature (°C)

Figure 2. Effects of Temperature on Heart Rate. The bar graph shows the mean heart rate of a
Rana pipiens at different temperature ranging from 4°C to 38.5°C.
 0.01
0.009 y = 0.0013x + 0.0015
R² = 0.9419
0.008
Heart Contractile Force (N)

0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
0 0.5 1 1.5 2 2.5
Distance of Stretch from the Reference Point (mm)

Figure 3. Effect of Tension on Heartbeat Force. The scatter plot depicted the contractile force of
a Rana pipiens heart when it is under different stretch distance. The reference point is 40.5mm
and it increases by an increment of 0.5mm. The line of best fit was added to the graph with its
correlation coefficient and equation.

58.8

58.6

58.4

58.2
Mean Heart Rate (BPM)

58

57.8
y = -0.0935x + 58.183
57.6 R² = 0.1778
57.4

57.2

57

56.8

56.6
0 0.5 1 1.5 2 2.5
Distance of Stretch from the Reference Point (mm)

Figure 4. Effect of Tension on Heartbeat Rate. The mean heartbeat rate was taken and graphed. It
shows the heartbeat rate relative to the stretch of a Rana pipiens heart. The reference point was
40.5mm and it increased by an increment of 0.5mm. The correlation coefficient was shown on
the trendline with the equation.
 0

-5
Percent Change in Heart Rate

-10

-15

-20

-25

-30
Drug Trials

Figure 5. Effect of Drugs on Percent Change in Heart Rate. The graph shows the percent change
in heart rate of Rana pipiens under different drug treatment. The drugs are Acetylcholine,
epinephrine, pilocarpine, atropine + acetylcholine.

0.6

0.5
Mean Time interval (s)

0.4

0.3

0.2

0.1

0
Normal Heart First-degree Block Second-degree Third-degree Block
Block

Figure 6. Effects of Conduction Block. Bar graph shows the mean time interval between the
atrial contraction and ventricular contraction of Rana pipiens’s heart. Each bar represents a
different heartbeat with different blockade. The Standard Error of Mean is also shown on the
bars.
Discussion

The study of Rana pipiens heart through this experiment helped us understand how

organisms like frog maintain a proper bodily functioning by adjusting the heart rate and

contraction frequencies. The significant of heart contraction starts with an action potential, and it

is generated by the heart cell, cardiomyocytes. The action potential allows ions like calcium

(Ca2+) or sodium (Na2+) to leak through their channels thereby depolarizing the cell (Luttgau et

al. 1958). This influence the Ca2+ to be released from the sarcoplasmic reticulum. The release

activates the actin-myosin reaction and shortens the sarcomere (Gordon et al. 2001). The result

shows the contraction of heart. Several different treatments were tested on the heart including

various temperature, stretch, drug, and different blockade on the heart.

Baseline. In the control experiment measuring the baseline, we got the data for mean,

maximum and minimum heart rate of a resting heart. The heart rate was recorded for 1 minute.

The mean for ECG channel was 19.353BPM and the maximum and minimum value was

198.601BPM and 6.952BPM, respectively.

Temperature. The hypothesise stated that that mean heart rate would decrease when the

heart was bath in 4 °C Ringer’s solution and increase when bathed in 38.5°C solution. Using the

room temperature of 21°C as a reference point (since it is very close to baseline), the heart in

cold bath beat slower (lower heart rate) than the heart in room temperature. The heart beat faster

when it was warmer than the room temperature. This allowed us to accept the hypothesis. Frogs

so not regulate their body temperature like we do because they are poikilotherms (Theodore

1955). Their body temperature various with the surrounding temperature changes (Hazel et al.

1974). This directly effects the number of cardiomyocytes that are active. When the surroundings

have a higher temperature, more cardiomyocytes were active therefore causes the heart rate
increased. As the increased in action potential in sinus venosus, the frequency of atrial

depolarization increased (Theodore 1955). The frequency was then transmitted to the ventricle

and generates contraction at a higher frequency. The low temperature decreased the transmission

of cardiomyocytes electrical signal to cause a reduction in heart rate.

Stretch. Another hypothesis stated that the heart contractile force increase as the stretch

increase. Also, the heart rate decreased after ventricular stretch of the heart. The original force of

contractile when the heart is relaxed was 0.0032N. The force increased up to 0.0094N when the

stretch was increased up to 2.5mm. The correlation coefficient and the regression equation both

shows a strong positive trend and therefore an increasing force of contraction when the stretch

was larger. The hypothesis was accepted. The explanation of this can be found in the Starling’s

law of heart, which stated that the force of contractile increase as the cardiac muscle stretch

increase (Stanley et al. 1954). It also stated that it reaches a maximum and decreases after the

maximum. This law dealt with the overlapping of actin and myosin mechanism in the muscle.

When the myosin is too crowded against the Z-disk at the end, the muscle generates zero or little

force because there is no more space to bring the Z-disk closer to each other (Gordon et al.

1966). As the muscles stretched out, the sarcomere length increases and the Z-disk moves further

apart allowing the myosin to be less crowded and therefore generates force when actin-myosin

shortens. The increase in sarcomere length also increased the sensitivity of the muscles to Ca2+

which generates a much stronger contraction (Gordon et al. 1966). The Z-disk now have enough

space to move towards each other to generate the contractile force. The further apart of the Z-

disk the greater force it generates when it shortens. This only goes to a point where the actin is

out of reach for myosin (Gordon et al. 1966). This is the point where the contractile force

decrease. The actin-myosin mechanism no longer works. The heart rate, on the other hand,
decreased as the preload increased. The negative correlation can be seen in figure 4. The

hypothesis was accepted. When stimulating a heart too rapidly, the Na+ and Ca2+ are overloaded

causing the heart rate to slow down (Matthew et al. 1984). When the heart is filled with blood,

the cardiomyocytes are stretched by the pressure in the ventricle (Stanley et al. 1954). When the

pressure is high, the stroke volume, which is the volume of blood the heart pumps out at each

stoke, increased. Therefore, as the stretch increased, the heart rate slowed down and the force of

contractile increased.

Drug. Drugs and transmitters can affect the heart contraction rate. Heart contracts due to

the depolarization from action potential. The release of Ca2+ from SR cause the actin-myosin

interaction to move the muscle (Xun et al. 2005). Drugs can speed up or slow down the pace of

heartbeat depending on the composition of the drugs. It was hypothesized that with the addition

of 0.1mg/mL of acetylcholine or 0.2mg/mL of pilocarpine decreased the heart rate. It is noted by

Bekker et al (1951) that acetylcholine and pilocarpine binds to receptors to reduce the action

potential in Daphnia thus decrease the heart rate. This proves our hypothesis correct.

Acetylcholine is a neurotransmitter that send signals to other cells. In the cardiac muscle, the

addition of acetylcholine increase the potassium outflux and decrease the Ca2+ and Na+ influx

of the pacemaker cells (Solange et al. 1981). This action decreases the depolarization rate and

increases hyperpolarization. The pacemaker rate now decreased, also slows down the heart rate.

Similarly, pilocarpine decreases the heart rate of the frog. Pilocarpine is a receptor agonist and

increases the activity of acetylcholine, which slow down the heart rate (Solange et al. 1981). On

the other hand, it was hypothesized that by adding 0.1mg/mL epinephrine and 1.0mg/mL of

atropine increases the heart rate. Unlike pilocarpine, atropine competes with acetylcholine for

receptors blocking it from releasing thus the heart rate increases. Epinephrine increases the Na+
and Ca+ influx into the pacemaker cell, increasing the depolarization rate (Hiroyuki et al. 1973).

This cause the heart rate to increase. The hypothesis was accepted. Our experimental data

however, shows that all drugs decreased heart rate. If correctly carried out the experiment, the

data for epinephrine and atropine should show an increase in heart rate. So, the percent change

should be positive.

Blockade. The final hypothesis stated that the time interval between atrial conduction

and ventricular conduction increased when the conduction blocks increased. The tightening of

the atrioventricular junction block the depolarization flow from atrium to ventricle The electrical

current flow through the gap junctions were blocked thus preventing the current to reach the

ventricular pacemaker cells (Bjurstedt et al. 1977). The first and second-degree block shows an

increasing time interval between the atrial and ventricular conduction. The third-degree block

was hard to observe because there was total constriction of current flow to produce contraction.

The time interval was the highest at the third-degree block showing a longer delay of the flow

from atrium to ventricle. The data was not completely reliable since the error bars do not over

lap. We cannot conclude that the data is significant. The constriction can cause death in

organisms as there will not be enough blood pumping to supply oxygen.

Conduct experiments on the frog heart under different condition allowed us to study the

behaviour of heart: the heart rate and force of contractile. The high temperature increased the

heart rate of the Rana pipiens and slowed down in a cool temperature. This is because they do

not have homeostasis to regulate body temperature, they rely on the external environment. The

force of contractile increased with the greater degree of stretch to a point and the heart rate

decreased with the greater stretch. The heart rate also varies with the addition of various drugs,

acetylcholine and pilocarpine decreased the heart rate while epinephrine and atropine increased
the heart rate. The blockade of AV junction decreased the time interval of atrial and ventricular

conduction as the blockade is stronger. Study of the blockade of Rana pipiens heart can relate to

human heart blockade. The different experiments can be conducted on the Rana pipiens heart

pacemaker cells to solve the blockade and maybe apply to human heart problem.
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Appendix
Table 1. Comparison of Resting Heart Rate
Calculated from Force Channel Calculated from ECG Channel
Mean Heart Rate (BPM) 68.051 19.353
Maximum Heart Rate (BPM) 288.428 198.601
Minimum Heart Rate (BPM) 17.443 6.952

Table 2. Effects of Temperature on Heart Rate

Mean Heart Rate (BPM)
Normal Salin (21°C) 64.157
Warm Salin (38.5°C) 81.496
Cold Salin (4°C) 46.504

Table 3. Effect of Tension on Heartbeat Force and Rate

Heart Contractile Force (N) Mean Heart Rate (BPM)
Basline (No Stretch) 0.0032 58.55 0
Stretch 1 (41mm) 0.0041 57.848 0.5
Stretch 2 (41.5mm) 0.0042 57.653 1
Stretch 3 (42mm) 0.0069 57.376 1.5
Stretch 4 (42.5mm) 0.0075 57.622 2
Stretch 5 (43mm) 0.0094 58.0863 2.5

Table 4. Effects of Drugs on Heart Rate

Heart Rate Before Heart RateAfter Drug Percent

Drug Administered Administrered Change in
(BPM) Heart Rate
Acetylcholine 25.258 18.117 -28.3
Epinephrine 31.142 28.731 -7.74
Pilocarpine 25.76 22.373 -13.1
Atropine+Acetylcholine 27.265 22.643 -16.9

Table 5. Conduction Block

Time Interval
Beat number Normal Heart First-degree Block Second-degree Block Third-degree Block
1 0.45 0.39 0.43 0.5
2 0.51 0.32 0.45 0.48
3 0.46 0.43 0.44 0.52
4 0.48 0.38 0.48 0.54
Mean 0.475 0.38 0.45 0.51
Standard Deviation 0.026458 0.045461 0.021602 0.02582
Sample Size 4 4 4 4
SEM 0.013229 0.02273 0.010801 0.01291