1. The Effects of Nitroglycerin on the Heart Rate of the 120-hour in vivo and in vitro Chicken Embryo Adrienne Dorward, Susan Mette, Jacqueline McLaughlin, Ph.D. The Pennsylvania State University, Berks-Lehigh Valley College

2. Introduction The development of the embryonic chicken heart begins with a series of cellular migrations, fusions, and specific differentiations. The heart develops from the fusion of a pair of precardiac mesodermal tubes that give rise to four regions located anterior to posterior: bulbus cordis, ventricle, atrium, and sinus venosus (McLaughlin and McCain, 1999). The 120-hour chicken embryo heart develops into 4 chambers: left and right atria, and left and right ventricles; at this time the sinus venosus has become the mature pacemaker or the SA node, which regulates the electrical signals that cause muscle contractions in the embryonic heart. http://www.lv.psu.edu/jxm57/chicklab/outline.html#introduction

3. Background Nitroglycerin (NG) is a common cardiac drug used to treat angina pectoris, and is also administered to prevent a myocardial infarction; this drug lowers blood pressure. It is well known that NG relaxes vascular smooth muscle, venous more than arterial. Diagram from www.chemfinder.cambridgesoft.com C3H5N3O9C3H5N3O9 Figure 1A: Chemical Structure of NG

4. Background The dilation of venous smooth muscle promotes peripheral pooling of blood, which results in a lower venous return, and the amount of blood that the heart has to pump results in a decreased left ventricular end-diastolic pressure. This decreases the workload on the heart and the amount of oxygen as well (Physicians’ Desk Reference, 2001).

5. Background When NG is administered in the body, enzymes within the cell convert it to the gas, nitrous oxide (NO). NO then functions as a hormone by stimulating an enzyme in the plasma membrane, which creates a second messenger. The second messenger relaxes nearby smooth muscle, which allows the blood vessels to dilate. In the heart, the dilation of the coronary vessels occurs. (Campbell, 2002).

6. Purpose We investigated the effects of Nitroglycerin (NG) on the in vivo and in vitro 120-hour embryonic chicken heart rate before and after exposure to 0.008%, 0.08%, and 0.8% NG concentrations. In a side study, we explored the effects of adding a 0.02% alcohol (Etoh) solution, a known vasodilator and depressant, after administering the 0.8% concentration of NG in both the in vivo and in vitro embryos.

7. Purpose We hypothesized that exposure to NG would cause bradycardia on both the in vivo and in vitro heart rates, directly proportional to the NG concentration, due to vasodilation of the coronary vessels and increase in cardiac output. The addition of Etoh would enhance the effects of NG by further inducing bradycardia, leading to fibrillation and cardiac arrest because both are known vasodilators.

8. Methods Prepared NG concentrations of 0.8%, 0.08%, and 0.008% from an 80% NG liquid stock and chick saline. An Etoh concentration of 0.02% was available for use. Obtained four, 120-hour chicken embryos/eggs. Employed Cruz’s (1993) “window” method. Determined in vivo heart rate for 15 seconds (5 times) for each. Added 2 drops of a separate concentration of NG to each embryo. Determined subsequent heart rates. Added Etoh to embryo #4. Determined its heart rate.

9. Methods Obtained three, 120-hour chicken embryos/eggs. Windowed the eggs using Cruz’s (1993) “window” method. Explanted the embryos using Cruz’s (1993) “explantation” method. Determined in vitro heart rate for each. Added 2 drops of a separate concentration of NG to each embryo. Determined subsequent heart rates for each. Added Etoh to embryo # 3. Determined heart rate.

10. Data Interpretation In all trials, a decrease in heart rate was noted after the specific NG concentrations were added for both in vivo and in vitro. A decrease in heart rate was noted after adding the Etoh in the in vivo embryo, but the heart rate increased in the in vitro embryo. Periods of bradycardia, fibrillation, tachycardia, and other arrhythmias were noted with all trials, except the 0.008% NG concentrations.

11. Results The average heart rate for chick embryo #1 in vivo was 138 bpm. The average heart rate after adding 0.008% NG was 96 bpm. The heart rate decreased by 42 bpm after NG was added.

12. Results The average heart rate for chick embryo #2 in vivo was 134 bpm. The average heart rate after adding 0.08% NG was 117 bpm. The heart rate decreased by 17 bpm after NG was added.

13. Results The average heart rate for chick embryo #3 in vivo was 146 bpm. The average heart rate after adding 0.8% NG was 120 bpm. The heart rate decreased by 26 bpm after NG was added.

14. Results The average heart rate for chick embryo #4 in vivo was 105 bpm. The average heart rate after adding 0.8% NG and 0.02% Etoh was 53 bpm. The heart rate decreased by 52 bpm after both NG and Etoh were added.

15. Results The average heart rate for in vitro chick embryo #1 was 106 bpm. The average heart rate after adding 0.008% NG was 90 bpm. The heart rate decreased by 16 bpm after adding the NG.

16. Results The average in vitro heart rate for chick embryo #2 was 64 bpm. The average heart rate after adding 0.08% NG was 36 bpm. The heart rate decreased by 28 bpm after adding the NG.

17. Results The average in vitro heart rate for chick embryo #3 was 89 bpm. The average heart rate after adding 0.8% NG was 58 bpm. The average heart rate after adding 0.8% NG and 0.02% Etoh was 84 bpm. The heart rate decreased by 31 bpm after adding the NG, but increased 26 bpm after adding the Etoh.

18. Conclusion Bradycardia did occur for all concentrations of NG in both the in vivo and in vitro embryos, which supports our hypothesis. The heart rate of the in vitro embryo decreased in direct proportion to the increase in NG concentration, but did not in the in vivo embryo. The discrepancy may be due to the irregularities in heart rate, such as fibrillation and other arrhythmias that were noted with the greater concentrations.

19. Conclusion The side study of adding 0.002% Etoh after the administration of the 0.8% NG solution produced mixed results. The heart rate of the in vivo embryo decreased significantly, however, the in vitro heart rate increased, but was still lower than the control. This increase may be due to the observed constant fibrillation that occurred after the administration of Etoh, which also supports our hypothesis.

20. Conclusion Overall, NG proved to be a vasodilator and decreased HR. The findings in this experiment correlate with the known effects that this drug has on the human heart. NG indirectly creates a secondary messenger that causes the dilation of coronary vessels. This vasodilation decreases the workload on the heart, lowers blood pressure, and ultimately results in a lower heart rate. In all trials, the heart muscles were relaxed at some point.

21. Future Experiments Use lower concentrations of NG to see if bradycardia will still occur. Use higher concentrations of NG to test if too much will induce cardiac arrest. Administer the Etoh before the NG to see if NG will increase the effects of Etoh for a side study. Use caffeine, a known stimulant, instead of Etoh to see if the caffeine would reverse the effects of the NG for a side study.

22. References McLaughlin, J.S. and McCain, E.R. “Development and physiological aspects of the chicken embryonic heart”. 1999. http://www.lv.psu.edu/jxm57/chicklab/(12 Feb. 2003). Physicians’ Desk Reference (55 th ed.). 2001. Public Medical Economics Co., Montvale, New Jersey,1604 pages. [ISBN 1-56363-375-2] [book]. Campbell, N.A. and Reece, J.B. Biology (6 th ed.). 2002. Benjamin Cummings, New York, 1038 pages. [ISBN 0- 8053-6624-5] [book].