Effects of Estrogen and Nitric Oxide Deprivation on

Effects of Estrogen and Nitric Oxide Deprivation on Late-Developing Zebrafish Heart Health Presented by: Brice Scott, Nicole Blixt, Jeff Nutter, Ian Mc. Farland, Derrick Ziglar, and Garrett Parsons BI-420 W-01 Seminar Research Presentation Biology Department Virginia Military Institute 1

Introduction • Nitric oxide (NO) is a small gaseous molecule that has been found to be an intracellular messenger for major functions of the body such as blood flow, platelet aggregation, and heart and neural activity. • There are three nitric oxide synthase (NOS) isoforms: neuronal NOS (n. NOS), inducible NOS (i. NOS), and endothelial NOS (e. NOS). • Cardiac neuronal nitric oxide synthase (NOS 1 or n. NOS) has been studied in depth in regards to regulating myocardial contraction and relaxation. It is done so by targeting specific calcium (Ca 2+) handling proteins, protein kinase-dependent or phosphatase-dependent phosphorylation/dephosphorylation and redox homeostasis. 2

Introduction Cont. • The N-nitrosylation pathway has been implicated in protecting the heart from arrhythmic behavior. • Deficient S-nitrosylation of the cardiac ryanodine receptor, Ry. R 2, has a variable effect on the sarcoplasmic reticulum Ca 2+ leak in isolated myocytes, possibly causing heart arrhythmias. • DTT is an inhibitor of the N-nitrosylation pathway. 3

Hypotheses • Both Estrogen and nitric oxide deprivation can: – Cause similar significant anomalies in latedeveloping zebrafish heart rates. – Be quickly corrected through AI treatment and n. NOSI washout. • Arrhythmias in 4 -6 day old embryonic zebrafish can be caused by the inhibition of the S-nitrosylation pathway. 4

Methods • Zebrafish were kept in 28. 5 C incubator and the ERS control medium was changed out every 24 hours. • Fish used younger than five (5) days post-fertilization were exposed to protease for dechoronation. – Fish 5 -7 days post-fertilization were not exposed to protease • Heart rates were measured by a stopwatch up to 30 seconds then multiplying that number by 2 to obtain a value. • The “listless” condition was determined by the lack of swim escape function when stimulated with a probe. • Arrhythmic values were determined to be heart rates below 120 beats/min. 5

• AI study Methods Cont. – Treatments: ERS and 50 u. M of AI – Fish heart rates were evaluated and noted every 24 hours – After 48 hours, all fish were washed out with ERS • n. NOSI dose response study – Treatments: 75 u. M n. NOSI and ERS control group – Fish were monitored for 48 hours post-treatment for “listless” condition and heart rates – After 48 hours fish were washed out with ERS to determine the rate of recovery – HRs and “listless” condition recorded every 24 hours 6

Methods Cont. • DTT experiment – Treatment groups: DTT— 25 u. M, 50 u. M, 75 u. M and ERS as a control • DTT is an inhibitor of the N-nitrosylation pathway – Fish were monitored and evaluated after the first hour and then additionally every 24 hours, for a two day period. – Heart rates, the listless conditions, and survivability were marked. • Data analysis: – T-test, z-test, and ANOVA were used 7

AI Experiment 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Avg. Heart Rate Vs. Treatment 150 * 75. 0% ERS AI * 15. 2% Heart Rate BPM % Listless Percent (%) Listless Vs. Treatment 140 * 128. 8 130 * 106. 1 120 ERS AI 110 100 Control Treatment 50 μM Fig 1 - Illustrates the percentage of zebrafish population displaying the listless condition in treatments of ERS and AI. Note: The AI treatment causes a significant increase (*=p<0. 05) in the “listless” condition when compared with that of the control. Control 50 μM Treatment Fig 2 - Exhibits the zebrafish populations average heart rate when exposed to a dose mediated treatment of ERS or AI. Note: The AI treatment significantly reduces (*=p<0. 05) heart rates when compared with that of the controls. 8

n. NOSI Experiment Percentage vs. Treatment Percentage (%) 120% 100% 80% 60% %survival 40% % listless 20% 0% ERS Control n. NOSi 75 μM Treatment Fig. 3 - Treatment at a concentration of 75 μM was analyzed after 48 hours beginning at 5 days post-fertilization. The percent survival and percent listless was calculated from all of the treatments. Note: n. NOSI treatment caused an increase in fish demonstrating the listless phenotype when compared to that of the ERS control group. 9

DTT Experiment Avg. Heart Rate Vs. Treatment Day 1 Heart Rate BPM 150 * 137. 5 140 133. 8 130 * 120. 3 120 ERS 110 DTT 100 Control 25 μM 50 μM Treatment 75 μM Fig. 4: Compiled dose-response data for day 1 of the heart rates (beats/min) in response to DTT treatment. Note: DTT treatment caused a significant decrease in heart rates at a 75 u. M concentration when compared to the control group (*=p<0. 05). 10

Conclusions • The data from the AI experiment demonstrated that: – AI treatment causes fish to display “listless” conditions within 75% of the population compared to 100% in the n. NOSI treatments. – A significant reduction in both heart rates and increased arrhythmias were observed similar to that of the n. NOSI treated fish. 11

Conclusions Cont. • The data from the n-NOSI dose-response and DTT experiment demonstrated: – DTT treatment significantly diminished heart rates and initiated greater arrhythmic values strongly indicating that the N-nitrosylation pathway is involved in this process. • This experiment also provided us with an ideal DTT dosage moving forward into the next phase of the experiment. 12

Help Received/Citations and Acknowledgments Help Received: Used information from Brice Scott’s research paper for introduction and COL Turner with revisions Citations: • Conti V, Russomanno G, Corbi G, Izzo V, Vecchione C, Filippelli A. 2013. Adrenoreceptors and nitric oxide in the cardiovascular system. Frontiers in Physiology. 4(321): 1 -11. • Cutler MJ, Plummer BN, Wan X, Sun QA, Hess D, Liu H, Deschenes I, Rosenbaum DS, Stamler JS, Laurita, KR. 2012. Aberrant s-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart. Proceedings of the National Academy of Sciences of the United States of America. 109(44): 18186 -18191. • Krause T, Gerbershagen MU, Fiege M, Weibhorn R, Wappler F. 2004. Dantrolene—a review of its pharmacology, therapeutic use and new developments. Anaesthesia. 59(4): 364 -373. • Zhang Y. 2013. Compartmentation of nitric oxide synthase regulates calcium signaling in the heart. Proceedings of the Physiology Society. 180 -181. • Zhao F, Li P, Chen SRW, Louis CF, Fruen BR. 2001. Dantrolene inhibition of ryanodine receptor Ca 2+ release channels: molecular mechanism and isoform sensitivity. The Journal of Biological Chemistry. 276: 13810 -13816. Acknowledgments: Thank you to the VMI Biology Department, COL Turner, Ms. Lozier, Cadet John Winalski ’ 16, and Cadet Vania Murcia ’ 17 for their help, critique, and advice this semester. 13