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An Examination of Extrasystoles and the Effects of Vagal Stimulation and Epinephrine on the Contractions of the Frog Heart

Nora Ko Group Cannon, Members: Daryl Chow, Alexandra Rochman, Chris Weber TA: Philip Matern August 6, 2012

Introduction
This experiment studied the bullfrog Rana catesbiana heart because if its close similarities with the human heart. The only difference between a human heart and a bullfrogs heart is that the bullfrogs heart contained two atria and only one ventricle. During the cardiac cycle, the oxygen poor blood returns to the right atrium via the sinus venosus, the region that corresponds to the vena cava in the human heart. Normally in the human heart, the two ventricles separate the oxygen rich blood from the oxygen poor. Most scientist would agree however, that the single ventricle in bullfrog do not mix the two types of blood, but that there are spongy folds called trabeculae that create two streams that separate the two (Bautista, Korber, 2009: p. 44). Additionally, there is a spiral valve within the conus arteriousus, which would be the aorta in the human heart, which helps direct leaving the heart to either the pulmonary or the systemic regions of the body (Bautista, Korber, 2009: p. 43-53). All in all, due to the close similarities, this study aimed to use the bullfrog heart as a paradigm to better understand the electrical and mechanical properties of the human heart, including extrasystolic contractions, the notion of vagal escape after bradycardia, and the use of epinephrine to drive a sympathetic effect in the heart. An extrasystolic contraction is basically a heartbeat different from the normal rhythm, and this often happens in normal people. It is caused by premature ventricular contraction caused by depolarization in the ventricle instead of the sinoatrial (SA) node (Sherwood 2010). The SA node is the main driver of an electrical impulse in the heart that generates the atria to contract, followed by the contraction of the ventricles. The term systole is used to describe the ventricles of the heart contracting to pump blood to the rest of the body, and this squeeze of blood causes the whole vasculature to be at peak pressure. The term diastole is used to describe the ventricles of heart relaxing and filling itself with blood from either the systemic (body) or pulmonary

(lungs) circulation. Thus, the rhythm of the heart, composed by heartbeats, is made up of a alternating pattern of systolic and diastolic activity in the heart. Hence, an extrasystole is an extra systole contraction that occurs in between heartbeats, causing a compensatory pause, or a time lag before the next normal ventricular contraction (Sherwood 2010). A time lag is different from the concept of bradycardia, however. Bradycardia is when the heart beats slower than its normal rate, and this is induced by a parasympathetic response, also known as the rest and digest mode, which is a physiological condition when a person is relaxed. On the other hand, the sympathetic response is when the body is very alert, almost to the point of panic; this is also known as the fight or flight mode. This phenomenon of how the heart beats more rapidly than normal is called tachycardia (Sherwood 2010). In the last part of this experiment, a solution of epinephrine will be dripped on the frogs heart in order to cause a tachycardia condition in the heart. The hypothesis of this experiment was that an extrasystole would induce a compensatory pause in the hearts beating rhythm, regardless whether the heart was stimulated during late diastole at threshold voltage, late diastole at two times the threshold voltage, or early diastole at threshold voltage. This compensatory pause would cause an increased amount of time before the next ventricular contraction and heartbeat. Additionally, an increasing voltage stimulation of the vagal nerve would cause the heart to become bradycardia, then cardiac arrest (stopping of the heart), and finally, a gradual increase in heart rate again. The heart-coming-back-to-life phenomenon was called vagal escape (Sherwood 2010). In the last part of the experiment, the prediction was that adding epinephrine would cause the heart to pump harder and therefore send more blood to the rest of the body.

Methods
The specific details of the procedures can be found in NPB 101 Physiology Lab Manual Second Edition (Bautista, Korber, 2009: p. 43-53). In this lab, we dissected a sedated bullfrog and isolated its heart and its vagus nerve. We occasionally bathed the frog with Ringers solution and massaged its muscles and organs throughout the experiment. Next, we burned off insulation of a piece of copper wire two inches from the wire end, and poked the wire through the ventricle of the heart. After twisting the short end to the long end of the wire a few times, we connected it to the force transducer and electrical cables. Once connected properly, we applied adequate tension by pushing the tray with the frog further from the transducer. We recorded the normal electrical and mechanical activity of the heart via the Biopac Student Lab software program and saved it under Heart Activity. Then, we set the stimulator to the settings indicated in the manual and switched start on the program. We switched the stimulator mode switch at late diastole, allowed ten beats to pass, and repeated this step two more times. Then, we repeated the last two steps with two times the threshold voltage, and again with stimulation at early diastole at threshold voltage. We recorded and saved the data after each trial. Next, we reconnected the electrical wires in preparation for vagal stimulation, and hooked the electrode to the vagus nerve. We determined the voltage for bradycardia by starting at 0.5V and slowly increasing until the heart rate slows down. We saved that data once again, restarted, and increased the stimulus voltage to 10-20% of the voltage used to cause bradycardia. Immediately after the heart arrests, we turned the mode switch to off and clicked stop. We waited until the heart resumed to a normal rate, and then recorded the baseline heart activity for another 30 seconds. We lifted the mode switch to Repeat and see the heart stop for a period, and we waited until the heart gradually began beating again at a lower rate. We allowed the heart to recover to normal, and

recorded the heart rate for two minutes before adding about five drops of epinephrine to the ventricle. We collected the data for six minutes and clicked stop to save the data.

Results
Extrasystolic Contractions The minimum voltage to bring about an extrasystole was 2.125V, with a corresponding tension of 0.87g. This force was slightly higher, but about the same as the force generated by a normal ventricular diastole, which was 0.79g. The time of a heart beat with an extrasystolic contraction in between took 3.03s. This was two times as long as a normal heart beat, which only took 1.50s. There was a delay time of 1.53s with the extrasystolic contraction. These values represent the extrasystole elicited upon late diastole and threshold voltage stimulation. When the threshold voltage was doubled to 4.25V, and the heart was stimulated at late ventricular diastole again, the tension elicited by the extrasystole was 3.99g. This is about four times the force generated at threshold voltage. In addition, this force was slightly lower, but like in the first scenario, it was about the same as the force generated by a normal diastole, which was 4.06V. The time of a heart beat with an extrasystolic contraction took 2.96s. This is also consistent with the first scenario, being two times the normal heart beat, 1.49s. The lag time was 1.47s. When the heart was stimulated during early diastole with threshold voltage, the force elicited by the extrasystole was somewhat higher than the previous scenario, 4.87g instead of 3.99g. It was also about six times stronger during early diastole than late diastole at the threshold stimulation. This extrasystole tension of 4.87g was about the same force as that created by a

normal ventricular diastole, 4.51g. The time of an heart beat with an extrasystole was 2.80s, about two fold of a normal heart beat, and so the time delayed was 1.42s, see Table 1.

Table 1. Extrasystole during late diastole at threshold voltage, late diastole at 2X the voltage, and early diastole are shown below with their relative forces, time of heart beat, and time of delay compared with those of a normal diastole. Extrasystole during Extrasystole during Extrasystole during Late Diastole with Threshold Voltage Stimulus Voltage (V) Force of Normal Diastole (g) Force of an Extrasystole (g) Time of a Normal Heart Beat (s) Time of Heart Beat with an Extrasystole Time of Delay (s) 1.53s 1.47s 1.42s 3.03s 2.96s 2.80s 1.50s 1.49s 1.38s 0.87g 3.99g 4.87g 2.125V 0.79g Late Diastole with 2X Voltage 4.25V 4.06g 2.125V 4.51g Early Diastole

Consistent in each of the three scenarios, the time of a heart beat with an extrasystole was approximately double the time of a normal heart beat, see figure 1.

Vagal Stimulation The normal heart rate was 43 beats per minute (BPM), and the corresponding voltage was 0.5 volts. The heart rate at bradycardia was 40BPM, which is slightly lower, and the corresponding voltage was 1.5V. At 1.625V, the heart arrested, and it slowly and gradually started beating again. The final heart rate after cardiac arrest was 39BPM, see table 2.

Table 2. The effect of vagal nerve stimulation on the bullfrog heart. As the voltage gradually increased, the heart rate went from normal to a much slower pace called bradycardia. At 1.625V, the heart stopped briefly (cardiac arrest), and gradually started beating again. This phenomenon is called vagal escape. Voltage Beats per minute (BPM) Normal Bradycardia Cardiac Arrest Vagal Escape 0.5V 1.5V 1.625V 1.625V 43 40 0 39

While the effect of increasing electrical stimulation to the vagal nerve to decreased in force in cardiac contractions and eventually caused vagal escape, the addition of epinephrine caused the heart contractions to increase in force.

The Effects of Epinephrine Before the addition of epinephrine, the ventricular region of the heart was contracting at a tension of 0.77g. The heart rate was 44.1 BPM. After adding epinephrine, the heart contraction force increased to 1.43g, and the heart rate also increased to 50.0 BPM, see Table 2.

Table 2. The sympathetic effects of adding epinephrine to the frog heart in terms of tension and heart rate. Epinephrine caused the heart to contract with a greater amount of tension, and an increased heart rate. Tension (g) Heart Rate (BPM) Before Epinephrine After Epinephrine 0.77 1.43 44.1 50.0

Hence, the heart contractions significantly increased in force during diastole, and looking at the raw data, the atrial contractions gradually fused more and more with the ventricular contractions after the addition of epinephrine as well. The atrial and ventricular contractions only gradually became separate entities after two minutes after the fusion, see figure 2.

Figure 2. The data showed the atrial and ventricular mechanical contractions distinctively before adding epinephrine. After the addition of epinephrine, the two contractions graduation merged together, as shown in the second row of data, and gradually, the atrial contraction separated from the ventricular contraction, but were still not as distinct as it was before the addition as shown in the first row.

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Discussion
A cardiac cycle is a sequence of mechanical and electrical events that repeat with every heartbeat (Sherwood 2010). Mechanically, there is a systole phase which is basically the contraction and emptying of the heart, and a diastole phase, which is the relaxation and filling of the heart. In systole, what happens is ventricular filling, in which the atrioventricular valves (AV values) open, and the semilunar valves close. Then, an isovolumetric contraction occurs, in which all valves close, and ventricular contraction causes an increase in pressure, but no change in volume (Sherwood 2010). In diastole, there is a ventricular ejection, where the semilunar valves open, and the ejection of blood causes an increase in pressure and a decrease in volume (Sherwood 2010). Finally an isovolumetric relaxation occurs, where all valves close, and the ventricles relax, causing a decrease in pressure, but no change in volume (Sherwood 2010). These contractions are induced by an electrical stimulation that runs through the pacemaker cells, which are all primarily led by the sinoatrial node (SA node), that of which drives the pace of the beating heart. This experiment found that the time it took for a heartbeat with an extrasystole was twice as long as a normal heartbeat. This is true for all three scenarios: extrasystole caused by an electrical stimulation at threshold voltage during late diastole, two times the threshold voltage during late diastole, and threshold voltage during early diastole. In addition, the force elicited by an extrasystole at two times the threshold voltage is four times greater than the forced elicited by an extrasystole at threshold voltage. An extrasystole is an extra premature ventricular contraction often caused by depolarization in the ventricle rather than the SA node (Sherwood 2010). Hence, the extra contraction will definitely cause a longer time in the heart beat, especially since there is a lag

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after the extrasystole. This approximately 1.50 seconds of delay is the compensatory pause, which is similar to a refractory period that occurs in muscles. During a compensatory pause, the atrial depolarization (P-wave) sent from the SA node (or sinus venosus of the frog) cannot reach the ventricle because it is in the refractory period (Sherwood 2010). During a refractory period, a second action potential (AP) cannot be trigger until the membrane recovers from the previous AP (Sherwood 2010). This period of time is called the effective refractory period (ERP), and this is equivalent to the absolute refractory period in nerves (Sherwood 2010). There is also a relative refractory period of which an AP can occur (Sherwood 2010). In a ventricular AP, there are four phases: phase zero is a rapid depolarization due to influx of Na+ mainly; phase one is a rapid repolarization from inactivation of Na+; phase two is the plateau which is caused by influx of Ca2+; phase three is the repolarization due to K+ efflux and Ca2+ channel inactivation; phase four is back to the resting membrane potential (Sherwood 2010). This experiment studied extrasystoles,which in some other sources, are called premature ventricular contractions, and in the study discussed immediately below, ventricular premature beats. This fairly recent study in the Clinical Cardiology Journal found that early and intensive lipid-reducing therapy can decrease ventricular premature beating, or non-sustained ventricular tachycardia, in patients with acute coronary syndrome (He et al., 2010). Thus, extrasystoles are not just a benign beating of the heart outside the normal contractions; it can be part of serious tachycardia conditions that translates to cardiovascular disease. Since this study found that reducing lipid can decrease the extrasystoles, the question to be asked is whether or not inducing parasympathetic or sympathetic activity will decrease the occurrence of extrasystoles at normal heart rates, which can be an interesting topic to pursue in future studies.

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The frog experiment also found that increasing the voltage when the electrode is hooked onto the vagus nerve will cause the heart to slow down (bradycardia) and eventually stop (cardiac arrest); however, not before long, the heart rate will resume to almost the rate it was before the arrest. This resumption of the heart beat phenomenon is called vagal escape. The rate that the pacemaker cells are acting in the heart is what determines the heart rate (HR), and the pacemakers cells in the heart are the SA node, the atrioventricular node (AV node), the bundle of his, and the purkinje fibers (Sherwood 2010). During continued stimulation of vagus nerve, there is a constant parasympathetic output to the SA node, which will cause bradycardia and eventually cardiac arrest (Sherwood 2010). Parasympathetic fibers travel along the vagus nerve to innervate the heart and primarily release the neurotransmitter of acetylcholine (Ach), which will bind to muscarinic Ach receptors and slow the pacemaker activity in the SA node to make the heart rate decrease (Sherwood 2010). In the AV node, Ach decreases conduction velocity, making it more difficult to depolarize neighboring cells to threshold, hence bradycardia and cardiac arrest. However, not all pacemaker cells are affected in the heart after a depolarization because electrical signals travel via gap junctions (Sherwood 2010). So the other pacemaker cells that do not get stimulated will take over control of the heart rate. Vagal escape takes place and the heart will generally beat again, but at a slower HR because the pacemaker activity will be set by the next fastest pacemaker cell (Sherwood 2010). An interesting study published in the Pharmacopsychiatry journal found that patients with a major depressive disorder had a distorted time perception when their vagus nerves were stimulated (Biermann, 2011). The study shows that parasympathetic effects of vagus nerve stimulation on the frontal lobe, and it suggests that it might modulate time perception in patients with major depressive disorder (Biermann, 2011). Since vagal stimulation slows down the heart

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rate, it will worsen the effects of patients with depression and cause them to have difficulty perceiving time as part of their symptoms. The outcome of our study also showed that the addition of epinephrine increased the force of the heart contraction by two times, which is consistent with our hypothesis. This increase in force is caused by sympathetic activity, contractility, and a concept called preload, which will be explained in detail. In addition, the heart rate was also shown to have an increase of six beats per minute after adding epinephrine, which is due to the effects of sympathetic activity. The sympathetic nerves normally release norepinephrine or epinephrine when a person is excited. These hormones bind to cardiomyocytes called 1-adrenergic receptors to increase HR (Sherwood 2010). Moreover, these hormones also cause an increase in the Ca2+ current allowing for greater contractions of the heart. When Ca2+ increases, the actin-myosin binding sites will increase, thus causing an increased contraction of the heart (Sherwood 2010). An increase of the contraction of the heart will cause an increase in stroke volume, or the amount of blood being pumped out from the heart to the rest of the body. This concept is called contractility, in which the force contraction will increase without increasing muscle length, but rather, the sympathetic activity will increase force contraction (Sherwood 2010). Another way to increase stroke volume (SV) is called preload, which is basically an increase in venous return, or blood returning to the heart (Sherwood 2010). According to the Frank-Starling Law, the heart will contract more force during systole if it is filled to a greater extent during the diastole of the previous heartbeat (Sherwood 2010). More filling equals an increase of end diastolic volume, which then leads to a greater potential to pump out more blood during a systolic contraction, and an increased SV.

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A study done in Japan in 2010 found that there is a sympathetic neural influence on bone metabolism in microgravity (Mano et al., 2010). In other words, the study found a significant correlation between muscle sympathetic activity and bone resorption in microgravity. Bone resorption in microgravity means a disintegration of bones in microgravity, and astronauts in space is an example of people experiencing microgravity. So the correlation would be that there is a regulatory effect of the sympathetic neurons which keeps bones from metabolizing and may keep a person from getting osteoporosis when they age. This interesting study shows the benefits of epinephrine in inducing a sympathetic effect (what we had examined in our study) and its help in regulating bone metabolism. This experiment was conducted on the beating heart of a live and sedated frog. It was conducted as a means to demonstrate the mechanical and electrical properties to give a better understanding of the human heart. There are many similarities between the bullfrog heart and the human heart, but it is worthwhile to do a comparison of the two to acknowledge the differences. Frogs have a three chambered heart, two atria and one ventricle, whereas the human heart is four chambered, contain two atria and two ventricles. The pacemaker structure is called the sinus venosus, while the human pacemaker is primarily the SA node (Bautista, Korber, 2009: p. 44). The frog heart also has a collection of cells that form a funnel between the atria and ventricle that has similar properties to the AV node. Frog hearts do not have a specialized ventricular conduction system, so no Purkinje fibers. Also, they do not have a developed sarcoplasmic reticulum, so the primary source of Ca2+ is extracellular. All in all, the study was a fairly good exemplar of the electrical and mechanical properties of the human heart.

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Conclusion
In conclusion, the study found that electrical stimulation of a bullfrog heart at threshold voltage during late diastole caused the elicitation of an extrasystole, which prolonged the heartbeat because of a delay called the compensatory period. The appearance of an extrasystole and its causal of a time delay were apparent when voltage was increased to two times the threshold voltage, and also the same when the stimulation took place during threshold voltage at early diastole. Moreover, the force of an extrasystolic contraction was quadrupled when the threshold voltage stimulation was doubled during late diastole. In addition, the study also found that increasing and continuous stimulation to the vagus nerve of the bullfrog heart caused bradycardia and then cardiac arrest. The result of this was because the parasympathetic fibers along the vagus nerve innervate the heart to release more Ach, which will bind to Ach receptors which slow the pacemaker activity in the SA node. Thus, the HR went down and eventually stopped. However, vagal escape caused the heart to start beating again, but only under the normal heart rate. This was because the pacemaker cells that were not affected gradually takes control of the heart rate once again. Lastly, another outcome of the experiment was that epinephrine increased the force of contraction in the heart due to the sympathetic effects of increased contractility and increased venous return, also known as preload. All in all, the experimental results and conclusions were consistent with our predictions.

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References
Bautista, Erwin and Julia Korber. 2008. NPB 101L Physiology Lab Manual Second Edition, pp. 43-53. University of Davis, CA. Biermann, T. 2011. Time Perception in Patients with Major Depressive Disorder during Vagus Nerve Stimulation. Pharmacopsychiatry. 44(05): 179-182. De Ferrari, G.M., Harry J.G.M. Crijns, Martin Borggrefe, Goran Milasinovic, Jan Smid, Markus Zabel, Antonello Gavazzi, Antonio Sanzo, Robert Dennert, Juergen Kuschyk, Srdjan Raspopovic, HelmutKlein, Karl Swedberg, and Peter J. Schwartz. 2011. Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure. European Heart Journal. 32: 847 855. Mano, T., Nishimura, N., Iwase, S. (2010). Sympathetic neural influence on bone metabolism in microgravity (Review). Acta Physiologica Hungarica. 97 (4): 354 -361.

Sherwood, Lauralee. Human Physiology From Cells to Systems. Belmont: Brooks/Cole Cengage Learning, 2010. Print. Xian-Zhi He, MD; Sheng-Hua Zhou, MD;Xin-Hong Wan, MD; Hai-Yu Wang, MD; Qing-Hua Zhong, MD; Jian-Fang Xue, MD. 2010. The effect of early and intensive statin therapy on ventricular premature beat or nonsustained ventricular tachycardia in patients with acute coronary syndrome. Clinical Cardiology. 34(1): 59-63.

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