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The Study of Cardiac Muscle Physiology of Rana Pipiens by Examining the Effect of Temperature, Stretch, Drugs on the Heart and Conduction Blockage

Name: Rafal Haleem Student Number: 214264071 Due Date: November 6th, 2017 Submitted date: November 6th, 2017 at 2:30 pm TA Name: Farnaz Mansouri Section #: 04

Abstract: The study was conducted to investigate the effect of temperature, stretch, drugs of the heart and conduction blockade on the contraction of the cardiac muscle of Rana Pipiens. Contraction is being tested in this experiment by using Rana Pipiens heart or also known as the frog’s heart. A measure of the contraction was observed by testing out different factors, such as three different temperatures of the Rana Pipiens ringer solution (40, 10 and 25 Celsius). Also by stretching the heart 0.5 mm from the baseline which was 45mm for about 5 trials, and the addition of different neurotransmitters such as (Acetylcholine, Epinephrine, Pilocarpine and Atropine + Acetylcholine) with Acetylcholine, Epinephrine and Atropine + Acetylcholine having a concentration of (1 mg/mL) and Pilocarpine having a concentration of (0.2 mg/mL). lastly a thread is wrapped around the hooked heart and its being tightened slowly to observe the heart conduction (Backx, 2017). The reason of this study is to test different factors that can manipulate the heart contraction of the organism Rana Pipiens. The hypothesis of this experiment is that high temperature, more stretching and drugs such as Epinephrine and Atropine would result in an increase in heart contraction of Rana Pipiens. After the application of the different temperature ringer solution the heart rate was higher when the warm 40 Celsius solution and was lower with the cold 10 Celsius ringer solutions was being added. It is also observed that the stretching of the heart from the apex increases the heart rate, due to more contraction. The effect of

the neurotransmitters on the heart rate depends on the drug that its being used, so acetylcholine tends to decrease heart rate, whereas Epinephrine and Atropine tend to increase heart contraction, but Pilocarpine can either increase or decrease the heart rate depending on the concentration that its present at. So as a result, the factors that were tested did have an effect on the contraction of the Rana Pipiens heart.

Introduction: Every part of the body has to undergo some sort of contraction in order to result in an action. The intention of this experiment is to observe the effect of some factors such as temperature, stretch, drugs of the heart and conduction blockade and its effect on the contraction of the heart of the Rana Pipiens. The heart of the Rana Pipiens is different from the mammals’ heart because it contains three chambered heart instead of four and the in the frogs the pacemaker cells are found in the sinus venosus, whereas in mammals it is found in SA and AV nodes (Backx, 2017). Change in the temperature of the ringer solution that is isotonic relative to the body fluid of Rana Pipiens will cause an increase in heart rate contraction when warm and decrease contraction when cold (Sevcencu, C., Ardelean, C., & Tarba, C, 2007). The stretch to the Rana Pipiens heart will cause an increase in the heart rate due to the tension that is being caused and that would result in more blood flowing in (Ter Keurs, H, 1996). different drugs have different effect on the rhythm of

contraction of the heart and they influence the pacemakers’ activity by having different polarization in the sinus cells (Hartzell, H, 1979).

The hypothesis that was made for this experiment was that increase in temperature will result in higher Rana Pipiens heart contraction, and increase in stretch will cause an increase contraction of the heart. The addition of the drug acetylcholine will slow the heart rate and the addition of Epinephrine, Pilocarpine and Atropine + Acetylcholine will increase the heart rate contraction, and this hypothesis was supported by different studies that were conducted by different researchers. A change in temperature of the ringer solution will have different effect on the heart contraction of the Rana Pipiens, so the effect of low temperature causes a decrease in the heart rate, and it will result in the highest rate of depolarization (Cedrini, L., & Alloatti, G, 1979). and another research that have been conducted about the impact of the stretch of the heart from the apex and its effect on the heart rate, and studies show that when a heart fails to generate any contraction this as a result a failure of cardiac cell to generate calcium ions, and the mechanical work that results from the heart will be diminished and results in the heart to fail (Ter Keurs, H, 1996). Methods that should be followed to test my hypothesis is the setup of the Lab Chart softwhere, by calibrating the force transduces. Then the frog that is used in this experiment which is Rana Pipiens has to be dissected around the heart area. Then after a hook is attached to the apex of the heart and a tension is caused to pull the heart upward using the force transducer, and measurements is then taken when different factors is being tested (Backx, 2017). The factors that were tested had different

affect such as hot temperature can increase the heart rate whereas cold results in a decrease. Stretch of the heart will result in an increase in heart rate due to more flow of blood, and drugs such as acetylcholine will result in a decrease in heart contraction, and opposite of that are epinephrine, Pilocarpine and Atropine increases the cardiac muscle contraction of Rana Pipiens. Methods and materials: Please refer to animal physiology I, Biol 3060 Lab Manual for the methods that was followed for the conduction of this experiment (Backx, 2017). Results: The experiment was designed to investigate some factors such as temperature, stretch, drugs of the heart and conduction blockade on the contraction of the Rana Pipiens heart. So, testing for contraction was obtained by the change in temperature using three different ringer solutions, one that was normal saline at 25oC, one that was Warm Saline at 40oC and another that was cold saline at 10 oC.

Table 1: Effects of three different states of temperature on the Heart Rate (BPM) of Rana Pipiens. Mean Heart Rate (BPM) Normal Saline (25) oC

52.8213

Warm Saline (40) oC

71.3612

Cold Saline (10) oC

37.3995

800000 700000 600000 500000 400000 300000 200000 100000 0

Normal Saline (25) oC

Warm Saline (40) oC

Cold Saline (10) oC

Figure 1: The effect of different temperatures of ringer solution, normal (25°C), warm (40°C) and cold (10°C) on the frog Rana pipiens mean heart rate (BPM). Figure 1 results were plotted in a bar graph, and it shows the relationship between the different temperatures of the ringer solutions and the mean heart rate (BPM), and this figure was contracted using the information from Table 1. Different results were obtained, as Figure 1 shows that the highest mean heart rate was as a result of the warm ringer solution and it was 71.3612, and the lowest heart rate was with the cold ringer solution and the result was 37.3995. Whereas heart rate in the normal ringer solution was 52.8213. In Table 2 the results were to measure both the force of the heart contractile in (N) and the mean heart rate in (BPM) with respect to the stretch of the heart by 0.5mm for 5 times and the baseline was 45mm. So, Figure 2 was obtained to show the relationship between the heart contractile force and the mean heart rate. And the result shows that the heart contractile force when from the baseline to the first stretch increases from 0.0058 to 0.0077, whereas the mean heart rate decreases in the first stretch from 48.9915 to 48.7812. from the 1st to the 2nd stretch the force

increases from 0.0077 to 0.0085 and so as the mean heart rate from 48.712 to 50.1869 (BPM). for stretch two to three the force increases from 0.0085 to 0.0116, and a decrease in the mean heart rate from 50.1869 to 48.1936 is observed. The force the decrease from stretch three to four and increase from stretch four to five, whereas the mean heart rate decreases all the way from stretch three to five as figure 2 shows. Table 2: Effect of Tension on Heartbeat Force and Rate Heart Contractile Force Mean Heart Rate (BPM) (N) Baseline (No Stretch) 0.0058 48.9915 Stretch 1 (45.5) mm

0.0077

50.7812

Stretch 2 (46) mm

0.0085

54.1869

Stretch 3 (46.5) mm

0.0116

55.1936

Stretch 4 (47) mm

0.0122

56,5150

Stretch 5 (47.5) mm

0.0135

58.1258

Figure 2: The effect of the ventricular muscle stretch on the strength of contraction for the frog Rana pipiens cardiac muscle

Table 3 investigates the effect of different drugs such as Acetylcholine, Epinephrine, Pilocarpine and Atropine + Acetylcholine on the contraction of the heart rate of Rana Pipiens heart. So, in Table 2

different results were recorded for each drug that was used in the experiment, and that includes the heart rate before drug administrated, heart rate after drug administrated and the percent change in heart rate, that was calculated using the rate with drug minus the resting rate and divided by the resting rate, and all that multiplied by 100 to achieve the percentage. As a result, Figure 3 is a bar graph of the percentage change in heart rate vs the drug administrated, and bar graph is used because the independent variable that is measure is qualitative and not quantitative. The Figure shows that the drug Acetylcholine has a negative percentage compare to the rest of the three other drugs that were added to the heart while contracting, and those are Epinephrine, Pilocarpine and Atropine + Acetylcholine. Table 3: Effects of Drugs on Heart Rate Heart Rate Before Drug Administered (BPM)

Heart Rate After Drug Administered (BPM)

Percent Change in Heart Rate

Acetylcholine

26.55

24.72

-6.89%

Epinephrine

29.6692

37.0619

24.91%

Pilocarpine

31.0628

35.0769

12.92%

Atropine + Acetylcholine

24.6496

32.1418

30.39%

Figure 3: The effect of different drugs on frog Rana pipiens heart rate. Discussion: The experiment was carried out to investigate some factors such as temperature, stretch, drugs of the heart and conduction blockade and its effect on the contraction of the cardiac muscles of Rana Pipiens. The study’s objective was to test contraction in the heart of Rana Pipiens and how the different factors has an impact on the contraction. Contraction is as a result of the ventricle muscle stretch, and the greater the stretch the greater the contraction, that is supported by starling law. The cardiac muscle contraction, in this experiment the heart of Rana Pipiens, when the heart rate slows down, the vagus slows so more blood can get in there, the more blood that get in, the more stretches and that results in a strong contraction. In table 4 in the appendix, Temperature has an effect on amphibians, because for example in this experiment the Rana Pipiens was studied, and so temperature in these organisms is not controlled, because their body temperature depends on the external environment. In figure 1 When the

warm ringer solution was added to the heart of Rana Pipiens, it resulted in an increase in heart rate by 71.3612, whereas the cold ringer solution caused a decrease in heart rate by 37.3995. the fact that there was an increase in heart rate after adding the warm ringer solution is because there was fast propagation of action potential which results in a reduce of cardiac muscle tension (Templeton, G, 1974). However, when the cold ringer solution was added there was a slow in propagation of action potential, which results in less heart contraction. The second factor that would have an effect on the heart contraction is the stretch of the heart. the ventricle increases in size because of the blood flow, then the heart rate contractile force increases (KobirumakiShimozawa, F et al, 2014). In the first and second contraction, there was an increase in heart contraction, and that was due to the high blood flow to the ventricle. And then third stretch to the fifth cause an increase in heart rate as well (Courtice, G, 1989). So therefore, the results show a positive relationship between the heart rate and contractile force. The heart contraction changes based on the amount of blood that can get in the vagus which results in more stretch, this is referred to as starling law. The higher the amount of blood that is present inside the heart the stronger the contraction that is generated (Courtice, G, 1989).

To determine the effect of drugs on the contraction of the heart, Acetylcholine and Epinephrine, Pilocarpine, and Atropine + Acetylcholine were used. The acetylcholine will be secreted by the vagus nerve and parasympathetic nervous system into the SA node, and therefore hyperpolarization of pacemaker cells will be resulted due to the increase of

the movement of potassium and decrease the movement calcium (Hartzell, H, 1979), and therefore the heart rate will decrease. In this experiment, the results of the addition of acetylcholine to the Rana Pipiens heart caused the heart contraction to slow down from 26.55 to 24.72. and these results agree with the hypothesis that was made earlier. Epinephrine effect on the heart rate will cause an increase in contraction because epinephrine is being released in to the blood stream by the medulla of adrenal gland, so the percentage change was 24.91% (Herman, C, 1983).

Pilocarpine results in an increase in acetylcholine receptors, which results in an increase in the release of acetylcholine (Ringer, S, 1881). This increase in heart contraction depends on the amount of concentration of Pilocarpine that is present (Alipov, N. N., Kositskiĭ, G. I., & Kuznetsova, T. E, 1985). In this experiment, the concentration of the Pilocarpine was (0.2 mg/mL), which resulted in an increase in cardiac muscle contraction in Rana Pipiens from 31.0628 to 35.0769 which had a percentage change of 12.92%. Atropine and Acetylcholine addition to the Rana Pipiens heart caused an increase in contraction from 24.6496 to 32.1418 with a percentage change of 30.39%, and that’s because Atropine blocks the acetylcholine receptors from releasing acetylcholine in to the blood stream so therefore the heart contraction increases (Woodbury, L, Hecht, H & Christopherson, A, 1951).

Appendix: Table 1: comparison of resting heart rate Calculated from Force Channel

Calculated from ECG Channel

Mean Heart Rate (BPM)

42.81

25.22

Maximum Heart Rate (BPM)

110.09

181.64

Minimum Heart Rate (BPM)

39.91

11.67

Reference:  Alipov, N. N., Kositskiĭ, G. I., & Kuznetsova, T. E. (1985). Action of pilocarpine on the normal frog heart and in pathology. Biulleten'eksperimental'noi biologii i meditsiny, 99(2), 169-171.

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Cedrini, L., & Alloatti, G. (1979). Effect of temperature on the electrical and mechanical activity of lizard ventricular myocardium. Bollettino della Societa italiana di biologia sperimentale, 55(14), 1362-1368.

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Courtice, G. P. 1989. Effect of Temperature on Cardiac Vagal Action in the Toad Bufo Marinus. J. exp. Biol. 149: 439-447.

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Hartzell, H. C. (1979). Adenosine receptors in frog sinus venosus: slow inhibitory potentials produced by adenine compounds and acetylcholine. The Journal of physiology, 293(1), 23-49.

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Herman, C. A., & Sandoval, E. J. (1983). Catecholamine effects on blood pressure and heart rate in the American bullfrog, Rana catesbeiana. General and comparative endocrinology, 52(1), 142148.

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Kobirumaki-Shimozawa, F., Inoue, T., Shintani, S. A., Oyama, K., Terui, T., Minamisawa, S., ... & Fukuda, N. (2014). Cardiac thin filament regulation and the Frank–Starling mechanism. The Journal of Physiological Sciences, 64(4), 221-232.

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Ringer, S. 1881. Concerning the Influence of Season and of Temperature on the Action and On the Antagonisms of Drugs. J. Physiol. 1: 115-124.

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Sevcencu, C., Ardelean, C., & Tarba, C. (2007). Electrical and mechanical effects induced by cold temperatures in the ventricle of isolated Rana ridibunda hearts. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 148(1), 196203

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Templeton, G. H., Wildenthal, K., Willerson, J. T., & Reardon, W. C. (1974). Influence of temperature on the mechanical properties of cardiac muscle. Circulation research, 34(5), 624-634.

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Ter Keurs, H. E. (1996). Heart failure and Starling's Law of the heart. The Canadian journal of cardiology, 12(10), 1047-1057.

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Woodbury, L. A., Hecht, H. H., & Christopherson, A. R. (1951). Membrane resting and action potentials of single cardiac muscle fibers of the frog ventricle. American Journal of Physiology--Legacy Content, 164(2), 307-318.

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York University. Fall 2017. Animal Physiology I, Biol 3060 Lab manual...