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LAB EXPERIMENT: UNDERSTANDING THE
MECHANICAL AND ELECTRICAL
PROPERTIES OF CONDUCTION,
CONTRACTION, AND AUTONOMIC
INTERVATION IN BULLFROG HEART
Sibin Mathews
TA: Drew Tilley
May 12, 2016
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I. Introduction
The cardiovascular system is made up of blood vessels, approximately 5 liters of blood, and
the heart which is the main focus of this lab experiment. Cardiac muscle cells which make up the
heartare striated and under involuntary control in which they are mechanically joined via
desmosomes giving the intercalated appearance and electrically coupled through gap
junctions(Sherwood 2010, pg.258). The heart maintains blood pressure, pumps blood throughout
the body, and helps remove waste and also transport nutrients and other important materials to
the tissues(Sherwood 2010, pg. 302).Interestingly, the heart has mechanic and electrical
properties due to the different types of cardiac cells called contractile and autorhythmiccells. The
contractile cells are responsible for the mechanical work while the autorhythmic cells act as
pacemaker cells that generate rhythmic action potentials for the contractile cells. Specifically,
autorhythmic cells rhythmically depolarize through what is known as funny current channels to
provide action potentials for cardiac contractions at the sinoatrial (SA) node which is the fastest
and thus the pacesetting node(Sherwood, 2007, pps. 308-309).So to explore the seemingly
effortless activations of the nodes, and successive contractions of the different regions of the
heart to ultimately pump blood, the bullfrog (Rana catesbiana) was used to understand the
different properties underlying heart‟s electrical and mechanic activity, heart‟s response to direct
ventricular input and also autonomic input.
Compared to the human heart, the physiological and the anatomical properties of the
bullfrog‟s heart are distinctly different. For example, the sinus venosus (SV) in the frog heart is
the primary pacemaker; however, in the human heart, it is the sinoatrial (SA) node that is the
primary pacemaker. Physiologically, humans with a developed sarcoplasmic reticulum, simply
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initiate contractions through calcium binding allowing myosin and actin to interact to form cross
bridge formation; however, frog hearts mostly acquiretheir large amount of calcium for their
contractions extracellularly. Anatomically, both species have two atrias, and human hearts
separate deoxygenated blood from oxygenated blood through the different compartments
including two ventricles and the septum in the heart.However, unlike human heart, frogs only
have one ventricle in which the presence of the trabeculae, an anatomical feature not present in
humans is used to compartmentalize deoxygenated and oxygenated blood.Frog heart also lacks
purkinje fibers that propagate action potential through the heart, and also lack bundle of his
which is used to transmit action potentials from the AV node to the speturm and to the ventricles.
Furthermore, human have coronary circulation to which the frog also lacks (Bautista et al.,
2009).
In this experiment, the physiological conditions of conduction, contraction, and autonomic
innervation will be experimentally examined in the double-pithed frog. To explore the
mechanical and electrical activity of the heart, the ventricle will be directly stimulated, and as a
result ventricular contractions will be the main focus of this experiment to explore the intrinsic
and extrinsic mechanisms governing contractions. For the first part of the lab during regular
heart activity, we expect ventricular contractions to be stronger and greater than atrial
contractions because ventricles pump blood to the entire systemic and pulmonary circulation
while atria only pumps into the ventricles (Sherwood 2010, pg.319). Worth mentioning for the
next part of the experiment is that excitations initiated from areas other than the SA nodecan
result in premature ventricular contraction (PVC) or an extrasystolic contraction (ESC). This
ESC can affect the end diastolic volume (EDV) and also stroke volume (SV), which combined
with heart rate is a product of cardiac output (CO) (Sherwood 2010, pg.321).So to explore these
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properties governing Frank Sterling‟s law of the heart, ESC was elicited in which the hypothesis
was that the contractions would vary depending on the time available for ventricular filling and
time of stimulation.(Hoffman et al, 1965). Other properties also explored in this lab include the
frog heart‟s response to autonomic nervous input. The autonomic nervous system which is
composed of the sympathetic and parasympathetic system innervates and plays an important role
in heart rate and the force of heart contractions. During vagal stimulation,when the
parasympathetic system is activated, heart rate is expected to decrease, termed bradycardia, due
to decreased nodal phase 4 causing slower nodal firings (Sherwood, 2007, p. 326), and force of
contraction is also expected to decrease because of (Lewis, 2001). However, activation of
sympathetic system is expected to increase heart rate, termed tachycardia, due to an increase
frequency and the propagation of the action potential, and also an increase in the force of
contraction (Anzola, 1956).
Materials and Methods
A detailed „materials and methods‟ instructions used in this experiment can be found in
the NPB 101L Systemic Physiology Lab Manual (Bautista 2009, pg.43-53). The subject used for
the experiment was the double-pitched bullfrog (Rana castebiana), and the electrical and
mechanical properties analyzed from the frog was with the use of the Bio-Pac program, version
4.1. The contractile force produced by the frog‟s heart was collected using the p-p tool, and the
beats per minute was collected using the BPM tool. Furthermore, the compensatory and latency
periods were measured using the delta T tool.
In part 1 of the experiment, the frog was safely cut into to access the heart, which was
then followed byidentification and isolationof the vagus nerve. Contributing to errors during this
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part was cutting other nerves leading to the heart, and also mishandling and overstretching of the
vagus nerve.Next, in part 2 of the experiment, the transducer was calibrated and a copper wire
was inserted into the ventricle and then connected to the newly calibrated force transducer. Then
the electrical signals were set upaccording to the lab manual (Bautista et al., 2009, pgs. 46-47).
Contributing to errors during this part was that the cooper wire was accidently inserted further
from the apex than instructed. Next, in part 3, tension was adjusted and a base heart rate and
heart contractile force was recorded for 2 minutes. For the purpose of the lab report, five data
points were randomly selected within the 2 minutes of control heart activity, in which mean and
standard deviation was measured for atrial and ventricular force of contraction, heart rate, and
latency between electrical and mechanical activity. During part 4 of the experiment,the objective
was to induce extrasystole (ESC) contractions. However, time constraints and lab mishaps which
included a nonfunctional computer in which the frog had to be re set up in another lab station
followed by difficulties acquiring observable biometrics in the Bio-Pac system prevented data
collection. As a result, data was obtained for late diastole, 2X threshold during late diastole, and
early diastole from a fellow lab group in which instructions are assumed to have followed the lab
manual (Bautista et al., 2009, pg. 49). Data was again analyzed using the Bio-Pac program,
version 4.1 where two data points each were collected for (pre, during and post extrasystole in)
late diastole, two times threshold during late diastole, and early systole. The two data points for
each step at each event was then averaged and standard deviated for lab purposes.
In part 5 of the experiment, the isolated vagus nerve was stimulated using settings from
the lab book (Bautista 2009, pg.51). Specifically, the the stimulus voltage was incrementally
increased to .8 V were we observed and recorded bradycardia. The data was analyzed and
recorded as 10 seconds before and 10 seconds after bradycardia was initiated. Five consecutive
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data points were respectively taken from both before and after, in which mean and standard
deviation was collected for the lab report. In the next step, in part 6, the stimulus voltage was
increased to past the value that was used to induce bradycardia,which was 1.2 V, were we
elicited and observed cardiac arrest. Five consecutive data points wereselected from each of the
different phases of this experiment that were averaged and also standard deviated for the
purposes of the lab report. The phases included; one minute prior to vagal stimulation, two
minutes during cardiac arrest, one minute after the vagal stimulation, and finally, at also two
minute after vagal stimulation. Finally, in part 6, the effect was epinephrine was
evaluated.Again, due to lab mishaps (which included a dying frog) and time constraints, data was
acquired from a fellow classmate that is assumed to have followed lab manual protocols. For lab
report purposes, three points each were consecutively collected and analyzed right before
epinephrine injection, and 3 minutes after when we saw the effect of the 4-6 drops of epinephrine
injection. Again, the three values from before and after were averaged and standard deviated for
analysis.
Throughout the experiment, ringer‟s solution was applied to keep the skin and heart moist
to allow cutaneous respiration, as well as for the calcium and sodium ions that are necessary for
heart activity. Furthermore, the frog‟s legs and body were frequently massaged in between
experiments to allow venous return to the heart.
Results
Part 1: Animal Preparation
During this part of the lab experiment, results were not obtainable, but this was a critical
and necessary step to isolate the frog heart which was used throughout the lab experiment.
Part 2: Preparation of the Frog Heart
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Again, no results were obtained during this part of the lab experiment, but this was
another vital part of the lab to correctly identify and successfully isolate the vagus nerve.
Part 3: Electrical and Mechanical Activity of the Heart
Data was collected for approximately two minutes to obtain and examine baseline values
of ventricular and atrial contractions, heart rate and latency during normal heart activity. As seen
in Table 1, the mean ventricular force of contractions during regular heart activity without any
stimulation was 0.436± 0.008grams while the mean atrial contraction was0.176± 0.009grams,
which almost a 150% decrease from ventricular force. The mean heart beat was measured to be
35.8± 0.988beats per minute. The average latency was measured to be 190± 15.2milliseconds.
Table 1: Mean cardiovascular parameters for baseline ventricular force, atrial force, average
heart rate and latency were measured under regular heart activity with no additional
stimulation.
Cardiovascular parameters
Mean Ventricular Force (grams)
Mean Atrial Force (grams)
Average Heart Rate (BPM)
Latency (msec)
Amount measured
0.43623grams
0.175917 grams
35.76187BPM
190.8 msec
Part 4 Extrasystolic Contractions
To analyze the effects of extrasystolic contractions on the heart, voltages were delivered
to the heart via a stimulator during late diastole, 2X threshold during late diastole, and early
diastole. As seen in Figure 1, the forces were recorded before extrasystolic contraction (preESC), at extrasystolic contraction (ESC), and after extrasystolic contraction (post-ESC).
For late diastole, the lowest voltage that elicited a extrasystole contraction, called
threshold voltage voltage (which is the lowest voltage that elicited extrasystole), was 2.00 V.
During direct voltage stimulus in late diastole, the pre-ESC force was measured to be 1.63 ±.059
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grams while the ESC was 1.79 ±.056 grams (fig. 1). Thus, the ESC was 0.16 grams more in
contractile force than pre-ESC,which demonstrates a contractile force increase of 9.80 %.
Between the ESC and the post-ESC, there was a compensatory pause of approximately 1.23±
.073 seconds (table 2), in which the post-ESC contractile force was measured to be 1.38±.023
grams, which was less than the pre-ESC (fig. 1).
Next, the threshold was increased to 2X the initial stimulus voltage used elicit threshold.
As a result, the 2X stimulus at 4V in late diastole gave a pre-ESC force of1.77 ± .024 grams
while ESC was measured to 2.43 ± .062 grams. The ESC resulted in .66 grams of more
contractile force than pre-ESC, thus demonstrating a contractile force increase of 37.0 %.
Between the ESC and the post-ESC, thecompensatory pause was approximately 1.25 ± .061
seconds (table 2), in which the the post-ESC contractile force was measured to be 1.42±.023
grams, which was approximately a .350grams, or 20.0 %decrease in of in contractile force
compared to the pre-ESC force (Fig. 1).
During early diastole voltage stimulation, the threshold voltage was measured to be 2.0V.
However, before stimulation at 2.00 V, the pre-ESC force was 1.46 ± .046 grams while the ESC
force was measured to be 1.63 ± .239 grams, which was a 0.17 grams increase in contractile
force when compared to pre-ESC, thus demonstrating a 12% increase in contractile force. The
compensatory pause between the ESC and the post-ESC approximately at early diastole was
measured to be 1.41 ± .054 seconds (table 2), in which the post-ESC contractile force was
measured to be 1.72 ± .307 grams, which was approximately an increase of 0.260 grams, or
18.0% contractile force when compared to the pre-ESC (fig. 1).
Table 2: Mean compensatory pause during late and early diastole in seconds.
Late diastole
1.21 sec
Early diastole
1.41 sec
8
3
2.5
Force of Ventricular Contration (G)
Pre
Extra
Post
2
1.5
1
0.5
0
Late Diastole 2V
Late Diastole (2X Threshold)
Early Diastole 2V
Event
Figure 1. Force of the pre ESC, at ESC, and post ESC for late diastole, 2x threshold of voltage
in late diastole, and early diastole via direct stimulation of the ventricle in the bullfrog heart.
Voltages used were 2V, 4V, and 2V respectively.
Vagal Stimulation
Part 5A: Vagal Stimulation: Bradycardia
To understand the relationship between the parasympathetic system and its effect on the
heart, the vagus nerve was directly stimulated via hook electrodes to elicit bradycardia at .8V
voltage. As seen in figure 2 and 3, heart rate and force of ventricular contractility was higher
before stimulating bradycardia, which were measured to be 39.1 ±0.866 BPM, and0.493± 0.019
grams of contractile force, respectively. During bradycardia, however,both heart rate and
contractility force of the ventricle was measured to be 33.3 ± 0.92 BPM and 0.197 ± 0.022 grams
(Figs. 2, 3). This change is roughly a 15.0 % decrease in heart rate, and approximately a 60.0 %
decrease in ventricular contractility.
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Heart Contractile Force (G)
0.6
0.5
0.4
0.3
0.2
0.1
0
10 seconds before
10 seconds after
Phase in Vagal Stimulation for Bardycardia
Heart Rate (BPM)
Figure 2. Effect of bradycardia eliciting vagal stimulation on ventricular contractile force. Force
of contraction measured ten seconds before and after vagal stimulation for bradycardia at a
voltage of .8V.
45
40
35
30
25
20
15
10
5
0
10 seconds before
10 seconds after
Phase in Vagal Stimulation for Bardycardia
Figure 3. Effect of bradycardia eliciting vagal stimulation on heart rate. Heart rate was measured
ten seconds before and after vagal stimulation for bradycardia at a voltage of .8V.
Part 5B: Vagal Stimulation: Cardiac Arrest
During this part of the lab, the vagal nerve was stimulated until the heart stopped beating,
which is a condition called cardiac arrest. Initially, the frog‟s heartbeat and contractile force
before cardiac arrest for one minute was at a mean of 37.1 ± 1.11 BPM, and 0.643 ± 0.151
grams, respectively (fig. 4). After stimulation at a voltage of 1.20 V, cardiac arrest occurred in
which heart rate and contractile force was measured to be zero (figs. 4, 5). This cardiac arrestwas
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observed to lastfor two minutes after which thefunction resumed again, but with a slower heart
rate and contractile force. The respective heart rate and contractile force one minute after cardiac
arrest was 3.02 ± 1.27 BPM, and 0.135 ± 0.124 grams (fig. 4). In which two minutes after
cardiac arrest however, the values increased further to approach that of baseline, with heart rate
at 26.9 ± 1.52, and contractile force at 0.526 ±0.112 (figs. 4, 5).
Heart Rate (BPM)
50
40
30
20
10
0
1 min before
2 min during
1 min after
2 min after
Phase in Vagal stimulation for Cardiac Arrest
Heart Contractile Force (G)
Figure 4. Effect of cardiac arrest eliciting vagal stimulation on heart rate at a stimulus voltage of
1.2V. Heart rates were measured one minute right before, during cardiac arrest, and one and two
minutes after vagal stimulation for cardiac arrest.
1
0.8
0.6
0.4
0.2
0
1 min before
2 min during
1 min after
2 min after
Phase in Vagal stimulation for Cardiac Arrest
Figure 5. Effect of cardiac arrest eliciting vagal stimulation on heart contractile force at a
stimulus voltage of 1.2V. Heart contractile forces were measured one minute right before, during
cardiac arrest, and one and two minutes after vagal stimulation for cardiac arrest.
Part 6: The Effects of Epinephrine
Finally, the effects of the sympathetic nervous system on cardiac function was examined
and analyzed. Again, heart rate and the contractile force before and after introducing epinephrine
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into the ventricle were measured. As seen in figure 6, the mean heart rate prior to injection of
epinephrine was 32.9 ±0.931BPM. After introducing epinephrine into the ventricle, the heart rate
increased to42.7 ± 1.77 BPM (fig 6). The force generated by ventricular contraction before
epinephrine injection was 0.257± 0.032 grams, however,after injection, there was an increase to
a mean of .434± 0.149 grams (fig. 7). Thus, heart rate and contractile force showed an increased
Heart rate (BPM)
by 33.4% and 68.9%, respectively.
50
45
40
35
30
25
20
15
10
5
0
Before
After
Epinephrine Injection
Figure 6. Effect of epinephrine on heart rate. Heart rates were measured right before injection
and then 3 minutes after when the effect of epinephrine was seen.
Heart Contractile force (g)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Before
After
Epinephrine Injection
Figure 7. Effect of epinephrine on heart ventricular contractile force. Contractile forces were
measured right before injection and then 3 minutes after when the effect of epinephrine was seen.
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Discussion
Electrical and Mechanical Activity of Heart
There are two types of specialized cardiac muscle cells, which called autorhythmic cells and
contractile cells. Autorhythmic cells are pacemaker cardiac cells that do not contract, but instead
are specialized for initiating and conducting action potentials (AP) for working cells, which are
the contractile cells (Sherwood 7th edition, p.309). These cardiac autorhythmic cells lie in the
sinus venosus in the frog, and in the humans lie in sino-atrial Node (SA node), the atrioventricular (AV) node, the bundle of his, and the purkinje fibers where theydisplay pacemaker
activity due to HCN non-specific cation channels that slowly depolarizes until threshold is
reached at whichthe membranefires an AP which spread throughout the heart to trigger rhythmic
beating of the contractile cells without nervous stimulation (Sherwood 7th edition, 309).
Specifically, these autorhythmic cells initiate and conduct AP that first depolarize the atria to
initiate atrial contraction and stimulate the AV node, which then conducts and activate signal
through the bundle of His and into the left and right bundle branches to the purkinje fibers which
in the end result in the depolarization and contraction of the ventricles. Interestingly, the SA
node in the human heart depolarizes roughly 80 times per minute. Thus, making the SA node the
fastest and the primary pacemaker of the human heart (Sherwood 2010, p.310). However, the
average beats per minute for frog‟s heart was observed to be about 30bpm, which is set by the
frog‟s primary pacemaker called the sinus venosus.
Physiologically, the first part of the electrical pacemaker activity is due to closure of the
potassium ion channels and the opening and influx of sodium. The second part consists of
opening the transient (T)-type calcium ion channels, which result in reaching threshold and in the
end depolarizing the cells. This then causes the rising phase of the action potential, which is due
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to the opening of long lasting (L)-type calcium ion channels that ultimately closes and ends with
the falling phase, which is due to opening and efflux of potassium ion channels (Sherwood 2010,
p.310). Notably, the current slowly conducts through the AV node to allow complete ventricular
filling in a process called AV nodal delay. The pacemaker potential then goes travels through the
ventricular myocardium via the Bundle of His and Purkinje fibers which finally results in
ventricular contraction, which pumps the blood out of the heart (Sherwood 2010, p.313).
However, unlike autorhythmic cells, spreading of the action potential in contractile
cardiac muscle cells is different. The action potential from the pacemaker cell causes a rapid rise
in the membrane potential in contractile cells due to the influx of sodium ions through voltage
gated sodium channels (Sherwood 2010, p.313). This ultimately results in contractile action
potential in which there is a brief period of repolarization caused by the closing of Na+ channels
and simultaneous limited efflux of potassium through voltage gated K+ channels. The
characteristic plateau phase in the contractile cells is the result of slow Ca2+ entry from L-type
calcium channels and simultaneous reduction in potassium efflux through voltage gated
potassium channels. This then causes the rapid falling phase, which is a result of opening of
voltage-gated K+ channels, but the resting potential is maintained through leaky K+ channels.
Ultimately, it is these action potentialsfrom the heart‟s working cells called contractile cardiac
muscle cells that cause contraction and relaxation in heart chambers (Sherwood 2010, p.314).
As this electrical activity occurs, simultaneously, the mechanical activity is also occuring,
but with a delay due to conduction transit time. First before depolarization of atria, the AV
valves are open and the ventricles that are relaxed are passively filled from high to low
contraction during a period called ventricular and atrial diastole. Once the blood fills in the right
ventricle via tricuspid valve, the atria receives action potential from SA node and undergoes
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depolarization were atrial systole occurs. This causes a contraction that allows extra blood to be
pumped in the ventricle through AV valve. This step dramatically increase ventricular pressure
and thus closing the valve. Next, the action potential is conducted down the AV node and
through conduction network of bundle of his and purkinje fibers causing depolarization and
isovolumetric systole of the ventricles, which is were ejection of the blood occurs. Ventricular
volume and pressure decrease because of the ejection of blood into the systemic and pulmonary
circulations, and once the pressure drops below that of the atria, the AV valves open again and
the ventricles passively fill again with blood from the atria until the depolarization of the atria
continuing the cycle (Sherwood 2010, pg.322).
During the first part of the experiment, the mechanical and electrical activity in
conduction and contraction of the cardiac cycle was observed. The latency, which is the time
between the onset of electrical activity and the beginning of ventricular contraction, was also
observed. This latency was measured to be 190 milliseconds (table 1). This is because the
ventricles don‟t contract immediately. As explained in the prior paragraphs, this is because it
takes time for the action potential to conduct down from the SA node to the AV node (due AV
nodal delay)and through the network of bundle of his and purkinje fibers to finally reach the
ventricle. As a result, it was understood that the cardiac cycle is made up of both electrical and
mechanical activity in which thecontractile cells of the atria and ventricles are coordinated in
such manner that they relax and contract based on the delay and application of action potential
from autorhythmic cells. Next, the data shows that the ventricular force of contraction was150%
stronger than atrial force of contraction (table 1). This is because ventricles supply blood to the
systemic and pulmonary circulation of the body compared to the atria, which requires far less
force to pump blood just to the ventricles.
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Part 4: Extrasystole Contractions
In part four of the experiment, extrasystolic contractions were produced by electrically
stimulating the ventricle during late diastole, two times threshold for late diastole, and early
diastole. Specifically, an extrasystolic contraction was observed at a voltage of 2V for late
diastole, which was unexpectedlyhigh when compared to early diastole (which was also 2 V) (fig
1). It was expected that late diastole to be lower in voltage to simulate because the cardiac cells
during late diastole are in a later stage of relative refractory period (RRP) in which the cellsare
closer to threshold and easier to fire (compared to early diastole were cell is further from
threshold) (Sherwood, 2007, p.316). This is different from absolute refractory period, called
effective refractory period (ERP) in which the cells are unable to be restimulated because of
inactivated double-gated sodium channels, regardless of how intense the stimulation is (Linhart,
1965). This unexpected high voltage at late diastole could have resulted due to a fatigued
bullfrog heart or simply an error in timing the stimulations of the heart.
Compared to skeletal muscle which can have graded contractions with increased motor
unit recruitment, safely so, cardiac cells do not grad, but act in an all or none contraction
(Sherwood, 2007,p.308). Upon increasing the voltage to 4 V during two times threshold for late
diastolic stimulation, the force of contraction during extrasystole increased from 1.79 grams to
2.43 grams, or 35.8 % compared to threshold of late diastole (fig 1). This was also unexpected
because an increase in voltage should not have affected the force of contraction during late
diastole. Regardless of voltage, unlike gradable skeletal muscle which can recruit motor units
with increasing voltage, cardiac muscle cells do not recruit or grade (Sherwood, 2007, p.308).
The results did not support this; and a reason for such discrepancy could have occurred from an
error in timing the stimulations.
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The ESC was also expected to contract with less force than post because it had less time
to fully fill with blood. Furthermore, The PESC was expected to have larger contractile force
because it had an increased compensatory period that allowed greater volumetric and pressure
filling which would have resulted in increased preload that should have resulted in greater PESC.
Under normal contractions, the cardiac muscle fiber‟s lengths are usually less that of optimal
length that is needed for maximum contraction (Hoffman et al., 1965). However, when there is
an increase in end diastolic volume through venous return and what is left in the heart because of
extrasystolic contraction, it is expected that an increase in cardiac muscle fiber length due to
increased pressure and filling would make the muscle fiber stretch more and cause a PSC that is
greater in force. (Sherwood, 2007, p.317). Both these predicated were proven wrong by the
collected data which could have resulted from a lack in venous return, which would justify the
decrease in in stroke volume, and decrease in contraction force contradicting Frank-Starling‟s
Law. In a previous study examining model fetal lamb hearts, the investigators were able to find
the end diastolic volume diameter of the left ventricle demonstrated a strong correlation to the
contractile force, tension values within the contraction of two-week-old hearts (Kirkpatrick et al.,
1976). Furthermore, In a study performed to understand Frank-Starling Law, an increase in
cardiac elasticity was observed among subjects along with an increased stoke volume to
accommodate for the increase in blood volume. However, in some of the subjects, when
contractile elasticity increased, a decrease in end diastolic volume was observed, in which the
researchers attributed the data to insufficient venous return (Chantler et. al., 2011).
As expected however, in all three types of stimulation, compensatory pause was
observed. A compensatory pause occurs because the cells are sent back into another absolute
refractory period due to the ESC. According to the Frank-Starling Law, this compensatory pause
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should allow for a greater time for filling resulting in an increased stroke volume and greater
force of contraction, and as a result, the force of contraction following the extrasystolic
contraction was also expected to be greater than the extrasystolic contraction. Cardiac output,
which is defined as the amount of blood pumped by each ventricle per minute, is determined by
two factors, which are heart rate and stroke volume.
Vagal Stimulation
a) Bradycardia
SA node is under tonic influence by both parasympathetic and sympathetic nervous system.
They bring about their effects on heart primarily by altering the activity of cAMP (cyclic
Adenosine Mono Phosphate) second-messenger system. The neurotransmitter acetylcholine that
is released form the vagus nerve binds to the muscarinic receptor which when bound couples
with an inhibitory G protein that reduces the activity of cAMP pathway. This decrease in cAMP
pathway ultimately causes an increase in the potassium ion permeability causing an influx of
potassium ions and thus making the cell negative than normal (Sherwood 2010, pg.326). As a
result, this causes the resting potential to start further away from threshold and harder to fire an
action potential which ultimately cause a decreases in the frequency of firing. The PNS
stimulation also decreases the excitability of the AV node which increases the AV nodal delay.
This AV nodal delay now result in the potential taking longer to conduct down the ventricles
through Bundle of His and Purkinje fibers. Therefore, during vagal stimulation as a result of both
a decrease in SA nodal firing, and an increase in AV nodal delay, heart rate decreases
(Olshansky, 2008).All these factors lead to decrease in cardiac output since cardiac output is a
function of heart rate and stroke volume. It was observed that the frog‟s heart rate reduced, a
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phenomenon called bradycardia, by 15.0% to 33.3 BPM (fig 3). Another effect of PNS activity is
that it weakens the contractility (fig 2). A research study done by Shumaker and team concluded
that a decrease in contractile strength is seen with vagal stimulation because Ach concentration
actually creates an negative inotropic effect that decreases CA concentration that is needed for
contractility (Shumaker, 1990). This can be seen through reduced ventricular force of contraction
according to the Frank Starling Law. In the experiment, ventricular force reduced by 60.0% to
.22 grams during bradycardia (fig. 2).
b) Cardiac Arrest/Vagal Escape
Cardiac arrest is a condition when the heart fails to contract. In frog, cardiac arrest was
achieved by continuously stimulating the parasympathetic vagus nerve in which both contractile
and heart rate decreased to zero for two minutes. AG Wallace and WM Dagget in the article
Pacemaker Activity during Vagal Escape Rhythms stated that cardiac arrest can be caused by
vagal stimulation either by the abolished activity of the SA node or by blocking the AV
conduction (Wallace, 1964). Thus, through vagal stimulation, the SA node and AV conduction
can be abolished to give rise to cardiac arrest; however, the data does not show tonic vagal
inhibition in which the heart during vagal stimulating first decreases and then drops to zero.
Instead, in lab we observed a rapid drop to zero which we analyzed as cardiac arrest.
One minutes after cardiac arrest, the heart rate and contractile force slowly resumed again.
The resumed values were a 3.00% increase in heart rate, and a .135 % increase in heart
contractility (figs. 4,5). This is because the frog‟s heart had undergone vagal escape in which the
cells from the AV nodes have taken over the function of Sinous Vinosus (which was the primary
pacemaker). In the experiment, Wallace and Dagget showed that vagal escape can occur from
pacemaker activity from AV node and below the Bundles of His (Wallace,1964). Two minutes
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after cardiac arrest, the heart rate had increased by 797% to 26.9 BPM and the contractile force
of the heart had increased to 270% to .526 grams (fig 4, 5). This is because the SA node is finally
beginning to resume its activity much like in bardycardia. Also, to maintain blood pressure, the
baroreceptors firing rate decreases (Sherwood 2010, pg.378). This leads to an increase in SNS
activity and decrease in PNS activity. Therefore, both these actions combined led to bringing the
blood pressure, heart rate, and contraction force closer back to baseline.
Part 6: Effects of Epinephrine
Compared to parasympathetic innervation, sympathetic nervous system works
antagonistically and innervates the ventricles, SA node, atria, and AV node. When sympathetic
nervous system is stimulated, norepinephrine is released and binds with -1 adrenergic receptors
that are coupled to a stimulatory G protein, which then activate the cAMP pathwaywhich results
in acascade that in the end result in increasing the sodium and calcium ion channel permeability.
Thischange in permeability results in raising the rate of spontaneous depolarization within the
SA nodal cell which now fire faster along with decreasing the delay in the AV nodal cells. As a
result, action potentials fire more frequently and propagate easier through the more excitable
conducting pathwaywhich contribute tothe faster heart rate. The data showed a of 33.4%
increaseto 42.7 BPM in heart rate, called tachycardia, after epinephrine injection (fig. 6)
(Sherwood, 2007, p.327).
As expected, contractile force of the heart also increased. This increase in contractile
force of the heart was measured to be a68.9 % increaseto.434 grams of contractile force
afterepinephrine injection (fig. 7). The contractile strength in the ventricles increased allowing
the heart to beat more intensely to eject a greater amount of blood volume from the left
ventricle.Sympathetic stimulations can also raise arterial pressure through an increase in cardiac
20
output and rapid ventricular ejections. (Anzola, 1956). This increase in contractility seen in
figure 7 can be explained by the inward movement of calcium through L-type calcium channels
which allow for more excitation contraction coupling leading to a greater contractile
force(Anzola, 1956). However, this increase in contractile force is regardless of the end diastolic
volume (EDV) (Sherwood, pg. 331).Compared to parasympathetic nervous system stimulation
were the pacemaker potential was longer and showed a decrease in slope; the pacemaker
potential in sympathetic nervous systemsimulation via epinephrine now has asteeper slope (that
is nowquicker to fire) (Sherwood, 2007,p. 327). Like the lab experiment, another study
performed by Morris Nathanson and Harris Miller, showed that epinephrine increased heart rate
and blood pressure in which they also claimed that these “sympathomimetic” compounds can be
used in cardiac therapy to aid in the prevention of cardiac arrest (Nathanson, 1952).
Conclusion
The purpose of this lab experiment was to examine the electrical and mechanical
properties of the cardiac muscle and ultimately the heart. First, the baseline activity of the heart
with no stimulus voltage was observed and recorded in which through, cardiovascular
parameters such as heart rate, contractile force, and latency were measured and understood.
Next, the ventricle was directly stimulated during periods of early diastole and late diastole to
acquire a better understanding of how interruption of the cardiac cycle affects the heart and how
the heart compensates for such disruptions. The effect of preload and afterload play significant
roles in affecting the cardiac cycle, which also are better understood in this lab. Ultimately by
referring to the first part of the lab of stimulating the ventricle directly, we are better able to
understand how stimulation and alterations of specific parameters affect stroke volume directly.
21
Once the intrinsic properties of the heart were explored, the extrinsic effects of the autonomic
nervous system were examined. First, the vagus nerve was stimulated in order to observe the
effects of the parasympathetic nervous system on the heart. Bradycardia, revealed with
increasing vagal stimulation, showed decrease in both heart rate and contractile force as expected
by the hypothesis. Next, with increased stimulation of the vagus nerve, cardiac arrest was
observed which also supported the hypothesis of very little to no heart rate and contraction.
Finally, the lab experiment concluded with the effects of the sympathetic nervous system on the
heart. The effects of the sympathetic nervous system were also observed accordingly to the
hypothesis which were increased heartrate and contraction. As a result, through these
experiments the intrinsic and extrinsic mechanisms governing cardiac contraction were examined
through electrical and mechanical activity of the heart. Furthermore, the understanding and the
knowledge gained from the electrical and mechanical properties of this lab experiment can be
further applied to numerous real-life contexts. For example, a large number of people due to
premature ventricular contractions suffer life threating heart arrhythmias. As a result, further
research into the heart is required to one day improve life and quality of patients with life
threating heart problems.
This lab experiment is funded and supported by the NPB 101 Lab curriculum under Dr. Erwin Bautista.
We also thank Drew Tilley, the teaching assistant, for the numerous discussions that contributed to this lab report.
22
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