Amphibious Mechanical and Electrical Cardiac Functions in Relation to the Human Heart

Miles Hirata Lab Partners: Brittany Gholson Hanna Hopper TA-Jacqueline Levin 11/16/12

1 Introduction The frog heart, although anatomically different from the mammalian human heart, most notably by having 2 atrias but only one ventricle, serves the same purpose of transporting blood, nutrients, and oxygen throughout the body and to eventually discard waste products. Blood is filled into the heart during periods of diastole, while systole refers to the pumping of blood out of the heart (Sherwood, 2010, p.321). The muscle contractions that cause the pumping action of the frog heart are stimulated by auto-rhythmic cells which elicit action potentials to cardiac muscle cells leading to their contractions. Auto-rhythmic cells of the frog heart are located in the sinus venosus, the cavity that receives blood from the veins and directs to the atria. Auto-rhythmic cells are characterized by having pacemaker abilities which dictate heart rate (Sherwood, 2010, p.309). Membrane potential of these pacemaker cells will continuously reach above and below threshold caused by influxes of sodium and calcium ions and an outward movement of potassium ions out of the cell (Sherwood, 2010, p.310). Cardiac contractile cells are stimulated by the pacemaker action potential via gap junctions, leading to their contractions (Haas et al, 1983, p.321). The duration of cardiac contractile action potentials, which can last 300 milliseconds, are relatively long-lasting when compared to the 1 ms of skeletal muscle action potentials. While cardiac contractile cells are above membrane potential threshold, no additional action potential can be formed. This area above threshold is known as the effective refractory period. As the cardiac myocytes membrane potential repolarizes below threshold back to resting membrane potential, a subsequent action potential can then be created by a strong enough stimulus, causing an extrasystolic contraction by the heart. This area of repolarization, leading to resting membrane potential is known as the relative refractory period. Part of this lab will consist of

2 attempting to generate a second cardiac muscle action potential before the prior action potential ends. My hypothesis states a second action potential will only occur during the relative refractory period of the first action potential of the contractile cell. While cardiac pacemaker cells control heart rate, stimulation of the sympathetic and parasympathetic nervous system can also have an effect on heart rate. Sympathetic stimulation results in increased heart rate or tachycardia, while parasympathetic stimulation of the heart via the vagus nerve leads to a decrease in heart rate, also known as bradycardia (Ng, 2001, p.319,329). Sympathetic stimulation causes nerves to release epinephrine or norepinephrine, leading to an increase of calcium and sodium influx, resulting in increased heart rate and contractile strength (Sherwood, 2010, p.327). Parasympathetic stimulation releases the neurotransmitter acetylcholine, causing an increase of potassium permeability of the pacemaker cells. This increase of potassium permeability hyperpolarizes the cell, making it more difficult to reach an action potential and can lead to cardiac arrest (Sherwood, 2010, p.326). If the heart reaches cardiac arrest due to vagal stimulation, cardiac contractions will eventually continue due to other pacemaker cells who are not as stimulated. This phenomenon is known as vagal escape. This experiment will observe the effects of parasympathetic and sympathetic stimulation on the heart. My hypothesis states heart rate will increase via sympathetic stimulation and decrease via parasympathetic stimulation. If cardiac arrest is reached, cardiac muscle contractions will eventually follow. The amphibious frog heart is adequately similar to the mammalian human heart which should allow these statements to hold true for both.

3 Methods All procedures pertaining to this experiment were taken from NPB 101L Systemic Physiology Lab Manual (Bautista, 2009, p.43-53). After plating the frog on a dissecting tray, exposure of the frogs thoracic cavity and heart was necessary in isolatin g the vagus nerve. Large bone scissors were used to cut the sternum medially, followed by the removal of the pericardium via forceps. The shoulder regions were pinned back in order to locate the vagus nerve, which was then tied to a thread for locating purposes. A double hooked electrode connected to the stimulator terminal was then placed in contact with the potential vagus nerve. Confirmation of the located vagus nerve was observed once the heart exhibited bradycardia from electrical stimulation by the electrode. Further vagal stimulation was observed until cardiac arrest and vagal escape were recorded. A copper wire was then used to puncture the ventricular wall, exiting the heart through the other side of the ventricle. The other wire end was tied to a force transducer at a 45o angle to measure force of cardiac contractions in grams with use of the Biopac software. Electrical connections, to produce extrasystolic contractions, were made with a spring clip, which was connected to the copper wire and stimulator. An injection of epinephrine was later used to simulate sympathetic activity on the heart. Throughout the exercise, water droplets were placed on the skin of the frog for continued respiration, while Ringers solution was used to saturate the heart for cardiac contractions to occur for the duration of the experiment. The frogs legs were also massaged in order to increase venous return to the heart. During the vagus nerve isolation, a proximal artery was severed, causing profuse blood loss, possibly having an effect on the data recorded. The vagus nerve also had to be isolated on the right side of the frog, as it could not be located on the left side.

4 Results The first experiment was to record the normal electrical and mechanical activity of the frogs heart. After the frogs heart was connected to the transducer via the copper wire, t he location and position of the frog was labeled with tape to attempt to standardize subsequent recordings of heart activity. The average value at systole peak, also corresponding to ventricular contraction, was 5.15 grams while diastole trough averaged 2.91 grams of force (Fig.1). Over the two minute recording, Electrical trace showed a continual slight decrease in activity.

Figure1. Normal electrical and mechanical activity of frog heart. Average ventricular contraction peak = 5.15g, average diastole trough = 2.91g.

ELECTRICAL TRACE

The following experiment attempted to create an extra cardiac contraction during both the early and late phases of diastole via electrical stimulation from the stimulator and

5 electrode. Early diastole was defined as the falling portion of the contraction recorded by the biopac software. Threshold to create an extrasystolic contraction during the early diastole phase was recorded at 2.9 volts of stimulation (Fig2). The late diastole phase was defined at the midpoint between two adjacent systole peaks. Threshold to observe an extrasystolic contraction at this location was 1.7 volts (Fig 3). Because of the use of the stimulator and electrode for this particular experiment, electrical activity of the heart was not recorded. The amount of electrical stimulus needed to observe an extrasystolic contraction was significantly higher during the early diastole phase relative to the late diastole phase (Table 1).

Extrasystolic C.

Fig 2. Extrasystolic contraction of frog heart during early diastole period. Smaller extrasystolic contraction followed by larger ventricular contraction. Threshold at early diastole phase=2.9 volts.

6 Extrasystolic C.

Fig 3. Extrasystolic contraction of frog heart during late diastole period. Threshold at late diastole=1.7 volts.

Table 1. Minimum amount of stimulus (voltage) needed to observe an extrasystolic contraction during late and early diastole phases in frog heart.
Threshold During Late Diastole 2x Threshold Late Diastole Threshold Early Diastole 1.7 volts 3.4 volts 2.9 volts

Voltage

Once the vagus nerve of the frog heart was isolated, a hook electrode was placed into contact with the nerve. Parafilm was used to block contact between the electrode and surrounding muscles. Baseline recording of the hearts beats per minute was valued at 38. Electrical stimulus of the vagus nerve was then increased until bradycardia was observed (Fig

7 4). During this time, heart rate showed a significant decrease relative to baseline heart rate (Table 2).

Figure 4. Comparison of mechanical and electrical activity of frog heart before and during bradycardia. Bpm at normal heart rate=38. Bpm during bradycardia=30.

Table 2. Beats per minute of frog heart before vagal stimulation, before bradycardia, and during bradycardia.
Voltage 0 volts 1 volts 1.8 volts (bradycardia) Beats per Minute 38 38 30

After bradycardia was observed, electrical stimulus was discontinued, allowing normal heart activity to return. When beats per minute returned to 38, the vagus nerve was then again electrically stimulated. The amount of stimulus was increased by 20% of the 1.8 volts that

8 induced bradycardia. At this stimulus value of 2.2 volts, cardiac arrest of the frog heart was first seen (Fig 5).

Figure 5. Mechanical and Electrical activity of frog heart at normal rate entering cardiac arrest via electrical vagal stimulation. Cardiac arrest threshold=2.2 volts.

When cardiac arrest was seen, electrical stimulus to the frog heart halted, allowing heart rate to recover to baseline. At this time, 2.2 volts of electrical stimulation continued, which caused the heart to enter cardiac arrest again. The electrical stimulus continued to stimulate the vagus nerve until cardiac contractions were recorded (Fig 6). During this time, beats per minute was recorded at a value of 12 (Table 3). Once vagal escape was observed, electrical stimulation ceased, allowing cardiac contractions and heart rate to return to normal.

Figure 6. Frog heart during cardiac arrest and entering vagal escape. 2.2 volts of stimulation via vagus nerve to induce cardiac arrest.

Table 3. Comparison of beats per minute of frog heart during baseline, electrical stimulation, and vagal escape.
Voltage 0 volts 1 volts 1.8 volts (bradycardia) 2.2 (cardiac arrest) 2.2 (vagal escape) Beats per Minute 38 38 30 0 12

While recording normal heart activity, epinephrine was injected into the heart through the ventricles. The outer surface of the frog heart was also saturated with epinephrine. Along with an increased heart rate and electrical activity, force of cardiac contraction increased as

10 well (Fig 7). After the epinephrine injection, beats per minute increased by 1.7, while electrical activity increased by 0.5 mV (Table 4).

Epinephrine injection

Figure 7. Mechanical and electrical activity of frog heart before and after injection of epinephrine.

Table 4. Maximum values and BPM of frog heart pre/post epinephrine injection. Increased activity after injection.
Max Value pre epi. Mechanical Activity 5.7 grams Electrical Activity 27.4 mV BPM pre epi. Max Value post epi. 43.9 7.3 grams 27.9 mV BPM post epi. 45.6

11 Discussion The results of the beginning experiment plots normal mechanical and electrical activity of the frog heart. The electrical activity reaches its highest value during the peak and falling phase of the large ventricular contraction wave. Because of the timing difference of action potentials between cardiac cells, depolarization of these cells will appear to last longer since the Biopac electrocardiogram measures the total electrical activity of the heart. The minimum value of electrical activity corresponds to atrial contraction, due to the ventricles containing significantly more cardiac muscle. Although the frog heart is fairly similar to the mammalian human heart, the frogs heart lacks calcium deposits from its sarcoplasmic reticulum and any calcium induced, calcium release function, obtaining calcium needed for contraction from extracellular space. Also, the frog heart must receive oxygen and nutrients from the lungs and through the frogs skin. Oxygenated blood from these sites return to the heart and mix with deoxygenated blood through the vena cava, dumping into the sinus venosus. Generation of extrasystolic contractions can only occur outside of the effective refractory period. The extended effective refractory period in cardiac muscle is attributed to the inactivation of sodium channels and the slow calcium influx during the plateau phase of the cardiac contractile cell (Sherwood, 2010, p.316). A long refractory period is desired in cardiac muscle as a precaution to prevent tetanus, or a single continuous contraction (Sherwood, 2010, p.316). Data from this experiment indicates the threshold to illicit an extrasystolic contraction during late diastole was 1.7 volts, while electrical stimulus threshold for early diastole was 2.9 volts. These values suggest cardiac cells are relatively easier stimulated during late diastole than early

12 diastole. This is in part due to early diastole being located in the relative refractory period of repolarization. At this point, the sodium channels that depolarized the contractile cells which lead to an action potential are mainly still in their closed and inactivated form (Sherwood, 2010, p.316). While the amount of stimulus needed to form an extrasystolic contraction changed with location, the results were similar. The small extrasystolic contraction was followed by a larger ventricular contraction. During the late diastole, the extrasystolic contraction measured at 2.75 grams of force, while the next ventricle contraction was recorded at 3.65 grams. The FrankStarling law illustrates the stretching of cardiac muscle will increase contractility, furthermore relating cardiac output to cardiac filling (Shiels, 2008, p.1). Due to the increased volume pumped out of the heart by the extrasystolic contraction, the amount of blood returned to the heart increased as well, shown in the increased ventricular contraction. The time between the extrasystolic contraction and the following normally produced contraction is known as compensatory pause and indicates a ventricular origin for an abnormal beat (Pritchett et al, 1980, p.1021). This pause allows for ventricular filling, allowing the increased volume of blood to be pumped into the ventricles. A compensatory pause is caused mainly by the extrasystolic contraction. During this contraction, the ventricles contract before atrial contraction, leading to a refractory period in which the heart is unable to continue normal contractions (Langendorf, 1953, p.401). My hypothesis was validated in the fact that extrasystolic contractions did occur during the relative refractory period.

13 The vagus nerve is the primary innervation of parasympathetic activity to both the human and frog heart. When stimulation of the vagus nerve began to increase, the frog heart experienced bradycardia at 1.8 volts of electrical stimulation (Fig.4). Beats per minute of the frog heart decreased from 38 to 30 during bradycardia, indicating tonic inhibition from the vagus nerve. Vagal stimulation causes the release of acetylcholine, which is suggested to slow the heart rate by suppressing diastole blood filling of the heart, from parasympathetic postganglionic axons (Bywater et al, 1987, p.35,36). (Also, the frog hearts mechanical activity in between contractions during bradycardia slightly increased from a minimum value of 2.09 grams compared to a minimum value of 1.94 grams during normal cardiac cycles, caused by acetylcholine acting on receptors to increase potassium conductance (Bywater et al, 1987, p.36). With regards to the human heart, parasympathetic stimulation decreases heart rate by increasing potassium permeability. (Sherwood, 2010, p.326). Parasympathetic stimulation also shortens the action potentials in the human heart, leading to a weakened contraction (Sherwood, 2010, p.326). However, this is also likely to occur with the frog heart. The electrical activity of the frog heart displayed a large repolarization phase towards the end of ventricular contraction during normal heart activity. During bradycardia, this electrical repolarization was replaced by a wide depolarization phase. This data suggests a reduction in refractory periods, caused presumably by decreased action potential times. After inducing bradycardia via parasympathetic stimulation, cardiac arrest was recorded at with 2.2 volts of vagal stimulation (Fig.5). When the frog heart experienced cardiac arrest, heart rate and electrical activity stopped, caused by the increased release of the neurotransmitter, acetylcholine. Increased vagal stimulation also indicates the membrane in a hyperpolarized state,

14 which makes action potentials difficult to occur. As cardiac arrest continued, there was an eventual slow but consistent heart rate caused by vagal escape (Fig.6). Heart rate measured in bpm during vagal escape was recorded at 12bpm. This drop in bpm is significant when compared to the baseline starting heart rate of 38bpm. Vagal escape is attributed to slower pacemaker cells, which are not inhibited by the vagus nerve stimulation, controlling the pacemaker ability. In the human heart, such pacemaker cells would be located in the Atrio-Ventricular node or purkinje fibers (Sherwood, 2010, p.310). Sympathetic stimulation of both the human heart and frog heart leads to a cell membrane depolarization (Bramich et al, 1991, p.403,404). For this experiment, epinephrine was used to simulate increased activity of the frog heart. Epinephrine, similar to adrenaline, was injected directly into the frog heart through the ventricles, and was also saturated with epinephrine. The epinephrine then acts on Beta-adrenoceptors of pacemaker cells, causing an increase in action potential (Bramich et al, 1991, p.404). As well as a 1.6 g increase in contractility, the epinephrine injection also increased bpm by 1.7. (Table 4). Such increasing values of contractility and heart rate stimulated by epinephrine are caused by a modulation of inward moving calcium (Bramich et al, 1991, 427). However, in a similar study with toads, these effects were not shown to simulate sympathetic activity. Instead of increasing amplitude of pacemaker action potentials and peak diastolic potential, only the speed of diastolic depolarization is changed (Bramich et al, 1991, p.427). While the data obtained does not accurately portray sympathetic stimulation, the outcome of increased heart rate is still shown. My hypothesis for both effects of parasympathetic and sympathetic was correct in determining the general outcome.

15 In conclusion, cardiac activity encompasses numerous systems and is governed by a balance of sympathetic and parasympathetic activity through the movement of ions and use of neurotransmitters acting on receptors. Not only is the functioning amphibious and mammalian heart complex and not fully understood yet, it also has integrated safeguards such as refractory periods and equal opposing forces which protects itself. Although the amphibious frog heart differs in a variety of ways from the human heart, results from this experiment can be used to understand how hearts of humans function through comparison and contrast.

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