1. EPINEPHRINE’S EFFECT ON AVIAN EMBRYONIC IN VITRO HEART RATE Aresh Ramin & Kelly Fetter

2. Purpose To determine the effects of epinephrine on the 4-day chicken embryonic heart rate in beats per minute (bpm). To investigate whether β -adrenergic receptors are developed in the 4-day chicken embryonic heart and their sensitivity to epinephrine. Previous research has shown that younger embryos, 2-2.5 days, contain less β -adrenergic receptor binding sites, where 5-day old embryos clearly have the β -adrenergic receptor binding sites, suggesting sensitivity to epinephrine (Lipshultz, 1981). Figure 1: Micrograph of 4- day old chicken embryo

3. Hypothesis The heart rate of the 4-day chicken embryo will increase linearly with the exogenous application of epinephrine and at toxic levels arrhythmias will be observed.

4. Basic Information The heart of the chicken embryo develops from the fusion of paired precardiac mesodermal tubes. Four distinct regions can be observed Conotruncus  aorta Ventricle Atrium Sinus venosus  pacemaker Blood flows anteriorly from sinus venosus to conotruncus. Atrium begins to expand in preparation for dividing into left and right portions (72 hours). Figure 2: 72-hour chicken embryo showing the development of the 4 chambers of the heart. http://www2.lv.psu.edu/jxm57/chicklab/outline.html#introduction

5. Adrenal Glands Triangle-shaped endocrine glands that sit on top of the kidneys Two distinct parts: Adrenal Cortex (Outer Core) Synthesis of corticosteroid hormones from cholesterol Adrenal Medulla (Inner Core) Synthesis of catecholamine hormones Norepinephrine Epinephrine Figure 3: Diagram of a adrenal gland located on top of a kidney http://www.merck.com/mrkshared/mmanual_home2/fg/fg164 _1.gif

6. Epinephrine Produced primarily from norepinephrine Administered by injection, inhalation, or topically to the eye A cardiac stimulant to increase heart rate and breathing Causes vasodilatation of blood vessels for muscles Increases psychological alertness Figure 4: Epinephrine being synthesized from norepinephrine in a synthetic pathway http://en.wikipedia.org/wiki/Image:Catecholamines_biosynthesis.svg

7. “Fight or Flight” The body's primitive, automatic, inborn response that prepares the body to "fight" or "flee" from perceived attack, harm or threat to survival. Initiated by the sympathetic nervous system. Allows the body systems to respond to stress. Figure 5: Molecular structure of epinephrine http://www.calstatela.edu/faculty/dfrankl/sbx2000/epin ephrine.gif

8. How does epinephrine work? The hormone epinephrine binds to the β -adrenergic receptor binding sites found in cardiac muscle cells. The receptors are linked to G -proteins, which are linked to adenylyl cyclase. Adenylyl cyclase converts ATP to cAMP (second messenger). The cAMP molecules are used to activate Protein Kinase A. Protein Kinase A phosphorylates the target proteins in the heart, which promotes Ca ++ output from the calcium channels. Muscle cells begin to contract once Ca ++ becomes available in the muscle cell cytosol and bind to the troponin. Epinephrine strengthens the heartbeat by mobilizing calcium.

9. Methods Five serial dilutions of a 1 mg/1 mL stock solution of epinephrine were made using CMRL media: 1 x 10 -5 mg/mL 1 x 10 -4 mg/mL 1 x 10 -3 mg/mL 1 x 10 -2 mg/mL 1 x 10 -1 mg/mL Five serial dilutions of a 1 mg/1 mL stock solution of epinephrine were made using CMRL media: 1 x 10 -5 mg/mL 1 x 10 -4 mg/mL 1 x 10 -3 mg/mL 1 x 10 -2 mg/mL 1 x 10 -1 mg/mL

10. Methods (Continued) Set up work station with necessary equipment. Kept goose neck lamps positioned in a way to keep the work area warm. Chose 4-day egg from incubator maintaining horizontal position. Carried egg to work station and placed in glass dish lined with cotton.

11. Methods (Continued) “Windowed” an egg according to the methods of Cruz, 1993: a. Placed Scotch Magic tape along the long axis of the egg and two more pieces of tape on either side of the center piece of the egg. b. Punctured the rounded end of egg with the tip of a scissor. Used a syringe needle to withdraw about 4 mL of albumen and emptied into the large beaker. c. Cut an oval opening using the punctured hole to expose embryo. d. Recorded the in vivo heart rate, the number of beats per minute (bpm), 3x’s at 15 second intervals each, into a table. Figure 6: Diagram showing a “windowed” chicken egg http://www.flickr.com/photos/egosumdaniel/305094250/

12. Methods (Continued) “Explanation” of the chicken embryo according to the methods of Cruz, 1993: a. Placed filter paper doughnut on blastoderm framing the embryo. b. Filled Syracuse dishes with CMRL and placed the dish on the stage of the dissecting microscope keeping it warm with gooseneck lamp. c. Cut vitelline envelope along edge of the doughnut with scissors. d. Lifted doughnut with forceps and embryo spoon to transfer embryo to the Syracuse dish. e. Removed all excess tissue from the chicken embryo. f. Recorded the in vitro heart rate (bpm), 3x’s at 15 second intervals each, into a table.

13. Methods (Continued ) Application of the drug a. Filled a warm Syracuse dish with the lowest concentration of drug. b. Transferred the embryo from the CMRL into the first drug concentration. c. Recorded the heart rate (bpm) after 1 minute, 3 x’s at 15 second intervals each, into a table. d. Used a pipette to remove the drug solution and transferred it back into the test tube. e. Transferred the second lowest concentration into the Syracuse dish. f. Recorded the heart rate (bpm) after 1 minute, 3 x’s at 15 second intervals each, into a table g. Repeated steps d-f for the remaining concentrations.

14. Controls In this experiment, two controls were used: In vivo heart rate of the embryos in their shells. In vitro “explanted” heart rate of the embryos in CMRL.

15. Chicken Embryo Heart Video

16. Results Figure 7: Histogram showing the average heart rates, in bpm, of all five experimental embryos prior to exposure to epinephrine (control data).

17. Results (Continued) Figure 8: Histogram showing the average heart rates, in bpm, for of all five in vitro experimental embryos exposed to various epinephrine concentrations.

18. Results (Continued) Figure 9: Histogram comparing the effect of various epinephrine concentrations on the heart rate, in bpm, of all five experimental embryos.

19. Conclusion The results of the experiment supported our hypothesis. As the concentration of epinephrine increased, the heart rate of the 4-day chicken embryo increased. The data shows that at the second highest concentration of epinephrine, 0.01 mg/mL, the heart rate was the fastest. Cardiac arrest resulted four out of five times at the highest concentration of epinephrine, 0.1 mg/mL. Arrhythmias such as atrial fibrillation, atrial flutter, and tachycardia were observed at the concentrations of 0.01 mg/mL and 0.001 mg/mL. The data suggests that the chicken embryo heart has the β - adrenergic receptors at 4-days or 96-hours, allowing sensitivity to epinephrine.

20. Future Research Use a larger number of experimental embryos. Conduct research in a controlled environment for the explanted embryos for stable conditions. Count the number of beats using time-lapse video microscopy and with this technology look closer at heart arrhythmias for proper diagnoses. Use older embryos in order to apply the drug directly onto the embryo while it is still in the shell. Embryos between 2 to 5-days will be stained with antibodies against the β -adrenergic receptors to validate their expression via immunofluorescence microscopy. Other cardiovascular drugs besides epinephrine will be tested on the chicken embryonic heart to compare/contrast effects on heart rate.

21. References Barry, A. (1950). The effect of epinephrine on the myocardium of the embryonic chick. Circulation. 1: 1362-1368. Cruz, Y.P. 1993. Laboratory exercises in developmental biology. Academic Press, San Diego, California, 241 pages “Epinephrine.” Gold Standard. 2007. http://www.clinciqalpharmacology.com (6 Mar. 2007) Lipshultz, S., Shanfeld, J.,& Chacko, S. (1981). Emergence of B- adrenergic Sensitivity in the Developing Chicken Heart. Cell Biology. 78, 288-292. McLaughlin, J.S. and McCain, E.R. (1997). In vivo and in vitro development of the chicken heart. Tested Studies for Laboratory Teaching, Volume 19 (C.A. Goldman, Editor). Proceedings of the 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE) 19: 331-332.

22. SPECIAL THANKS TO: Dr. Jacqueline McLaughlin Dr. David Dressler Nadia Abidi Dan Devine Steve Wentzel Soumya Immella Mrs. Eileen Grodziak Mrs. Stephanie Derstine Any Questions?