Effects of Temperature, Stretch, and Various Drug Treatments on the

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1 Nicole Rodi Bio 235: Animal Physiology Heart Muscle Lab Report 10/24/2014 Effects of Temperature, Stretch, and Various Drug Treatments on the Cardiac Muscle Activity of Rana pipiens Abstract Mechanical and electrical activity was monitored in a frog heart as it was subjected to various changes in temperature, stretch, and drugs treatment. A frog was dissected to expose its beating heart and hooked up to a force transducer, which was connected to Power Lab in the LabTutor software (LabTutor, AD Instruments). In separate procedures, heart rate and contractile force were observed by applying various temperatures of Frog Ringer's solutions to the heart, increasing degrees of stretch, and administering various drug treatments of Acetylcholine, Epinephrine, Pilocarpine, and Atropine. Increasing temperature of the Frog Ringer's resulted in an increase in heart rate. An increase degree of stretch resulted in an increase of contractile force. Acetylcholine and Pilocarpine slowed the heart rate when they were applied. Conversely, the addition of Epinephrine and Atropine + Acetylcholine increased the heart rate. These results demonstrate that heart rate and contractile force can be manipulated via various imposing factors used in this experiment, such as temperature fluctuations, increasing ventricular stretch, and drug administration, to monitor frog cardiac muscle activity.

2 Nicole Rodi 2 Introduction Beginning in the late 1800s, studies of isolated organs were first pioneered by scientist Sydney Ringer, who developed a solution (Ringer s solution) that could sustain an isolated organ from a pithed animal (Ringer, 1882). Typically, frog hearts were isolated, which will continue to beat with the addition of Ringer s solution for several hours allowing for the study of basic cardiac functions. The heart is made up of specialized tissue called cardiac muscle. Cardiac muscle contains myocardial cells, which consist of autorhythmic and contractile cells. The autorhythmic cells make up the sinoatrial (SA) node and the atrioventricular (AV) node, which are specialized pacemaker cells that spontaneously depolarize and repolarize; when funny channels open, the cells will depolarize to threshold, followed by calcium channels opening to depolarize the peak levels. Once the action potential has peaked, potassium channels open for a short amount of time, causing the falling phase known as the refractory period. The membrane potential falling below threshold then signals for the funny channels to open again and repeat the cycle. Firing of the SA node initiates this wave of depolarization, which spreads to the atria via electrical connections between cardiac muscle cells called gap junctions, causing the atria to contract. Eventually, the slow depolarization reaches the AV node, and then travels rapidly along the Bundle of His and Purkinje Fibers to the fibers of the ventricle. The slow-fast transmission of depolarization ensures the ventricles properly fill with blood and contract after the atria. This homeostatic process maintains a regular heartbeat (Weixel, 2014). In the leopard frog, Rana pipiens, control of heart activity is very similar to humans, making the frog heart a good model system. In general, the heart receives continuous input from the autonomic nervous system (ANS) and/or endocrine system, which release chemicals that

3 Nicole Rodi 3 alter both the rate and force of heart muscle contraction. The ANS is composed of two antagonistic divisions, called the sympathetic and parasympathetic nervous systems; the sympathetic division, often referred to as the fight or flight system, speeds up heart rate through Norepinephrine release onto beta-1 adrenergic receptors on the heart. The parasympathetic division, referred to as the rest or digest system, slows heart rate through Acetylcholine release onto muscarinic receptors on the heart. The endocrine system can also influence heart rate and force of contraction through the release of Epinephrine. Epinephrine binds to beta-1 adrenergic receptors, and causes a similar physiological response as norepinephrine (Silverthorn, 2012). Starling s Law of the Heart is another way to specifically explain force of contraction. It states that the greater the distention of the ventricle at the time of contraction, the greater the force produced, and thus the greater the volume of blood that is ejected (Silverthorn, 2014). In this experiment, the mechanical and electrical activity of the frog heart was monitored as it was subjected to various levels of temperature, stretch, and drug treatment. Through this study, cardiac muscle functioning can be observed and better understood, as well as understanding how the development of drug intervention for patients suffering from various heart conditions is becoming a reality. We predict that as the temperature of frog Ringer s solution is increased, heart rate will increase. Also, as stretch of the heart is increased, the force of contraction will increase as well, based off our knowledge of Starling s Law of the Heart. The drugs that will be used in this experiment are epinephrine, acetylcholine, atropine, and pilocarpine; we further predict that heart rate will increase after administering epinephrine and atropine to the frog heart, while acetylcholine and pilocarpine will decrease the heart rate.

4 Nicole Rodi 4 Methods Frog Dissection: The frog dissection and subsequent observations were aided by those of Johaner (2010) in the Dissection of a Frog exposing its beating heart video on Youtube.com, and Weixel (2014) in the Heart Muscle Lab Manual. A leopard frog was double pithed and dissected in order to expose its beating heart as described in the Heart Muscle Lab Manual (Weixel, 2014). The frog was faced ventral side up, and a cut was made through the pectoral girdle. The pericardium was then removed from the exposed heart, followed by placing a small hook, attached to the force transducer, through the apex of the ventricle. To keep the heart moist and active, the heart was continuously bathed with Frog Ringer's solution. The recipe for the Frog Ringer s solution is described in the Heart Muscle lab manual (Weixel, 2014). Originally, the frog was incorrectly double pithed, which caused the frog to move around as the dissection was performed. In the future, experimenters conducting this lab should properly double pith their frog specimen before proceeding to the dissection. Recording of Frog Resting Heart Rate: Using the recording procedure described in the Lab Tutor software, the force transducer was calibrated using a binder clip. The recording electrodes were attached to pins placed in the frog s limbs to monitor the mechanical and electrical activity on the ECG as the frog heart was subjected to various imposed conditions (LabTutor, AD Instruments). Once the transducer was calibrated, the recorded resting heart rate of the frog served as the baseline heart rate. Recording of Temperature Effects:

5 Nicole Rodi 5 To examine the effects of temperature on heart rate, the frog heart had been bathed in succinct in a cold (4 C), room temperature (27 C), and warm (37 C) Frog Ringer s solution, as described in the Heart Muscle Lab Manual (Weixel, 2014). The heart contractions were recorded for 30 seconds, with the addition of each temperature change, to be used for later analysis. Starling s Law of the Heart: Following the procedure outlined in LabTutor, along with instructor assistance, the degree of stretch of the heart muscle was carefully adjusted on the force transducer (AD Instruments) in increasing 0.5mm increments. The effect of increasing stretch on force of contraction was subsequently observed. Recording of Various Drug Treatment Effects: To record the effects of adding various drugs on frog heart rate in LabTutor, Acetylcholine, Epinephrine, Atropine + Acetylcholine, and Pilocarpine were applied in the order described by the Heart Muscle Lab Manual LabTutor (Weixel, 2014). The effects each drug had on the frog heart rate were noted; after Acetylcholine was on the frog heart for 20 seconds, the heart went into cardiac arrest, thus room temperature Frog Ringer s solution, along with four drops of Epinephrine, were applied to bring the heart back to a steady rate. Results and Discussion Effects of Temperature: To test the effects of various temperatures on the frog heart, the frog heart had been bathed in succinct in a cold (4 C), room temperature (27 C), and warm (37 C) Frog Ringer s solution. In

6 Nicole Rodi 6 cold (4 C) Frog Ringer s solution, the average heart rate (BPM) decreased, compared to the room temperature (27 C), and warm (37 C) Frog Ringer s solution. The average heart rate (BPM) in room temperature (27 C) Frog Ringer s solution was comparatively lower than that of the warm (37 C) Frog Ringer s solution. These values are represented in Table 1 and Figure 1. The effects of temperature found on heart rate (BPM) in this experiment are similar to those results found by Gillian Courtice (Courtice, 1989), whereas toad body temperature is lowered, the cardiac vague nerve becomes more effective at slowing the heart. The increased vagal effectiveness at low temperatures could be caused by a decline in cholinesterase activity, which would allow a greater build-up of ACh, and thus a more pronounced effect of the nerve at the pacemaker (Courtice, 1989). A two tailed t-test was created using the raw data collected from Group 1 and Group 2 comparing the effects of temperature on heart rate. Using the raw data (Table 2), the calculated t-value, , is less than the two tailed t-critical value, , therefore we would fail to reject the null hypothesis that there are any differences between the two sets of data. This is a positive outcome because it was expected that the temperature effects on the heart would be similar for each person conducting this experiment.

7 Nicole Rodi 7 Group 1 Group 2 Group 3 Temperature Heart Rate Heart Rate Heart Rate Average Heart ( C) (BPM) (BPM) (BPM) Rate (BPM) Cold Room Temp Warm Table 1. Thermal effects on frog heart rate (BPM) after bathed in cold (4 C), room temperature (27 C), and warm (37 C) Frog Ringer s solution. Temperature ( C) Heart Rate (BPM) Group 1 Heart Rate (BPM) Group 2) Table 2. Raw data collected form Group 1 and Group 2 on heart rate as subjected to various temperatures of Frog Ringer s solution.

8 Mean Heart Rate (BPM) Nicole Rodi Temperature ( C) Figure 1. Average temperature effects on frog heart rate (BPM) after bathed in cold (4 C), room temperature (27 C), and warm (37 C) Frog Ringer s solution. Effect of Stretch on Force: To test the effects stretch and force, the degree of stretch of the heart muscle was carefully adjusted on the force transducer (AD Instruments) in increasing 0.5mm increments. The data in Fig. 2. show the mechanical effects of increasing the degrees of stretch on force of contraction of the heart. At each degree of increasing stretch, the average contractile force (N) increased in a near linear fashion. This finding parallels Starling s Law of the Heart, which again states that the greater the distention of the ventricle, the greater the force produced. In terms of the cellular and molecular mechanisms at play to explain the finding that increased stretch increases contractile force, more blood will stretch the walls of the ventricle, thus increasing volume, and activating more crossbridges and calcium release in the heart muscle. The sarcomeres also stretch, which

9 Contractile Force (N) Nicole Rodi 9 increases the distance that actin and myosin can travel, producing a greater contractile force of the cardiac muscle Degreee of Stretch Figure 2. Starling s Law of the Heart demonstrated on the frog heart muscle, showing the effects of increasing stretch on the average frog heart contractile force (N). Effect of Pharmacological Intervention: As Acetylcholine, Epinephrine, Atropine + Acetylcholine, and Pilocarpine were applied to the frog heart in the order described by the Heart Muscle Lab Manual LabTutor (Weixel, 2014), Acetylcholine and Pilocarpine generally slowed the heart rate and Epinephrine and Atropine + Acetylcholine increased the heart rate. With the application of Acetylcholine, a heart rate of 55.2 BPM was recorded as dropping to 33.1 BPM. For Epinephrine, the heart rate increased from 33.3 BPM to 93.6 BPM. Administering Pilocarpine caused the heart rate to drop from 66.3 BMP to 41.8 BPM. Lastly, Atropine + Acetylcholine increased the heart rate from 54.0 BPM to BPM (Table 3, Figure 3). Using the raw data (Table 4) to see if there was a difference between

10 Nicole Rodi 10 heart rate before or after drug exposure, the absolute value of the calculated t-value, , is less than the two tailed t-critical value, , therefore we would fail to reject the null hypothesis that there are any differences between the two sets of data. Based on our prior knowledge of the drugs effects on parasympathetic and sympathetic control of the heart, we can assume that this counter evidence was due to chance or error. In general, Acetylcholine produces parasympathetic effects by binding to muscarinic cholinergic receptors, which activates G- proteins and opens potassium channels (hyperpolarization), while closing sodium and calcium channels. This caused the rate of action potential firing to decrease, thus decreasing heart rate. Epinephrine is a hormone secreted by the adrenal medulla together with norepinephrine, and acts to increase both the strength of contraction of heart rate by binding to beta-1 adrenergic receptors (Silverthorn, 2012). In 1881, scientist Sydney Ringer first experimented with Atropine and Pilocarpine to observe the effects on heart rate. Ringer had discovered that Atropine increased heart rate, while Pilocarpine decreased heart rate (Ringer, 1881). Atropine is an Ach antagonist, which blocks the muscarinic Ach receptors and effects of Ach, thereby increasing pupil size (dilation) and increasing heart rate (Animal Physiology Lab Lecture Notes, 2014). Lastly, Pilocarpine is a muscarinic receptor agonist that increases the activity of muscarinic acetylcholine receptor, increasing the effects of acetylcholine in the body. Since pilocarpine increases the activity of the parasympathetic nervous system, it slows down the heart rate (Silverthorn, 2014).

11 Nicole Rodi 11 Drug Treatment % Change in Heart Rate (BPM) Acetylcholine Epinephrine Pilocarpine Atropine + Acetylcholine 74.4 Table 3. Average pharmacological effects of various drugs on the heart rate (BPM) of the frog. Drug Treatment Group 1 Heart Rate (BPM) Before Exposure Group 1 Heart Rate (BPM) After Exposure Acetylcholine Epinephrine Pilocarpine Atropine + Ach Table 4. Raw data collected form Group 1 on heart rate (BPM) as it is exposed before and after various drug treatments.

12 Nicole Rodi 12 Figure 3. The graph above shows the average pharmacological effects on the frog heart rate (BPM) through the addition of various drugs treatments. Conclusion Recording the effects of various imposed conditions on heart rate and contractile force of the frog heart allowed for the basic understanding of cardiac function and mechanism. A linear relationship between increased temperature and increased heart rate was observed. To manipulate heart rate via drug administration, epinephrine and the combination of atropine and acetylcholine increased heart rate, however, epinephrine operates through endocrine mechanisms, while atropine + acetylcholine operate through neural mechanisms. Acetylcholine and Pilocarpine decreased the heart rate, which physiologically makes sense because ACh and

13 Nicole Rodi 13 Pilocarpine are both agonists to one another. Also, Starling's Law of the Heart was simulated through stretching the ventricle at the apex of the frog heart to increase force of contraction. To further expand our knowledge in this experiment about heart muscle, we could also manipulate ion concentrations such as decreasing or increasing sodium concentrations in the Frog Ringer s solution and see how this would affect heart rate and contraction. For example, too much or too little sodium can affect changes in the amount of water in the blood because sodium draws in water. Also, decreased sodium levels lowers blood volume, causing the blood pressure to drop and the heart rate to increase. Sodium increases raise blood volume, decreases heart rate, and causes fluid to accumulate, forcing your heart to work harder. These effects would be interesting to view on a frog heart for further cardiac muscle study and understanding. Acknowledgements I would like to thank my Tuesday Animal Physiology Laboratory colleagues for sharing their data, which allowed us to compare results, thus better supporting our interpretations of the findings in this experiment.

14 Nicole Rodi 14 References Courtice, G. P Effect of Temperature on Cardiac Vagal Action in the Toad Bufo Marinus. J. exp. Biol. 149: Ringer, S Concerning the Influence Exerted by each of the Constituents of the Blood on the Contraction of the Ventricle. J. Physiol. 4: Ringer, S Concerning the Influence of Season and of Temperature on the Action and On the Antagonisms of Drugs. J. Physiol. 1: Silverthorn, D.U. PhD Human Physiology: An Integrated Approach, 6th ed. Pearson Education, Inc Weixel, K Heart Muscle Lab Manual. BIO 235 Laboratory Handout. Washington & Jefferson College, Washington, PA.

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