TRPing up the Balance of Oxidative Stress - Transient Receptor Potential Vanilloid 1’s Role in Diabetic Microvascular Disease A Dissertation Presented in Partial Fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies, Northeast Ohio Medical University Daniel J. DelloStritto B.S., Integrative Life Sciences, Kent State University Integrated Pharmaceutical Medicine Northeast Ohio Medical University 2016 Dissertation Committee: Ian Bratz Ph.D. (Advisor) Altaf Darvesh Ph.D. William M. Chilian Ph.D. Marc S. Penn M.D., Ph.D. Derek S. Damron Ph.D Copyright Daniel DelloStritto 2016 Abstract Diabetes is a growing epidemic worldwide leading to an increase in cardiovascular morbidity and mortality. The maintenance of coronary blood flow (CBF) is crucial to supplying energy and oxygen to the working myocardium and this has been demonstrated to be disrupted in diabetes. Specifically, our lab has illustrated the ion channel Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is a key regulator in CBF control, but it’s contribution to CBF regulation is attenuated in diabetes. A crucial mechanism behind pathologies seen in diabetes is the imbalance in oxidative stress (OS). Herein, we determined the role for oxidative stress, specifically H2O2, to regulate TRPV1-mediated CBF—demonstrating H2O2 differentially regulates TRPV1. Acutely, H2O2 increases CBF through a TRPV1-dependent signaling; however, exposure to prolonged OS blunts TRPV1-mediated endothelial signaling resulting in uncoupling of CBF. Similarly, this altered OS environment is known to lead to an increase lipid peroxidation (LPO). The LPO product 4-hydroxynonenal (4-HNE) increases oxidative post translational modification (PTM) resulting in altered protein function. As such, we established the ability of 4-HNE to modify TRPV1 resulting in blunting of TRPV1- dependent CBF regulation. Further elucidation determined this process occurs via a carbonylation reaction on Cysteine 621 of TRPV1. Overall, this dissertation demonstrated a role for TRPV1 to sense the oxidative environment and subsequently regulate CBF; however, in diabetes this perfusion is perturbed through the oxidative modification of TRPV1 leading to potential avenues for future therapeutic targeting. ii Acknowledgments Working towards the ultimate goal of obtaining a Ph.D. can be quite treacherous, there are so many people along the way that impact your development as a scientist and as a person. I realize this list is long, however I believe each and every person truly contributed to this project either scientifically or keeping me in a balanced state. Without family as the backbone I would not be here today. I want to thank my mother and father for their support through the years. They have guided me in numerous ways. They have sacrificed and pushed me every step of the way and for that, I owe them a true debt. My journey down this road would not have started if not for Drs. Eric Soehnlen and Soumitra Basu, who have been there since day one. Dr. Basu, you allowed me the opportunity to find a passion I did not knew existed within me. Eric, working with you inspired me and opened my eyes to a world of science; I am truly thankful for all of your guidance through these many years in all aspects, not only as scientific sounding board but as a confidant. Finally, I need to thank my wife Jessica for whom I would not have completed this process without. The support needed to fulfill these requirements can be grueling and without this support I would not have been able to be as successful as I have been. During this process of professional growth there are those who have helped in other as aspects of my development. I would like to thank all of those friends whom I have made within the American Physician Scientists Association, you helped me grow as a physician-scientist and as a leader (thank you the BOD and EC of the 2015-2016 academic year including Drs. Alexander Adami, Dani Daye, Brittany Weber, Peter iii Mittwede, Evan Noch, M. Kerry O’Banion, Moshe Levi, and Michael Guo). To my collaborator and friend Karen Doserch, thank you for pushing scientifically and challenging me to remember the immune system. Also, I would like to thank Angela Bennett for her unending help in the design of the cartoon figures. Scientifically, I have been as lucky as I have personally. I want to thank the members of my committee for their countless hours of work and guidance through the years. Dr. William Chilian for providing leadership to the department as well as your thought provoking and crazy ideas. Dr. Derek Damron thank you for always pushing me to question our work and others. Also, I always looked forward able to the many light stories you told to provided that comic relief when necessary. Dr. Marc Penn for his advice about the many options open for physician-scientists. I want to thank the many other faculty members here at NEOMED (Drs. Charles Thodeti, Altaf Darvesh, Werner Geldenhuys, June Yun, Denise Inman, Yeong-Renn Chen, Yoon-Kwang Lee and Kenneth Rosenthal) that have helped my development as a student and a scientist. Also, I need to extend my gratitude to the fellow graduate students from IMS: Danielle Janota, Patti McCallinhart, Suzanna Logan, Rituparna Ganguly, Ravi Adapala, Dan Luther, Soumyadip Sahu, Holly Capelli and from Pharmaceutical Science: Gina Wilson and Matt Smith. Furthermore, I am grateful for those that I have had the honor of training: Brittany Klarich, Joe Fahmy, Sai Korada, and Pat Connell. You have always kept me on my toes and made sure to instill a sense of teaching and purpose into the science. Last, I have to thank Pritam Sinharoy and Spencer Andrei from the Damron lab for always engaging in challenging iv each other science. You helped expand my technical skills, but more importantly we grew scientifically in our many meetings pushing each other to justify our results or experiments. Next I need to thank those who have helped me develop my wide array of my technical expertise. I first must thank Drs. Greg Dick and Ibra (Drew) Fancher for expanding my horizons in the field of patch-clamp electrophysiology: it truly is as much of an art as it is a science. Bethany Prudner, I have to thank you for teaching me about molecular biology and sparking a passion within me. Crucial to furthering more intricate designs in the molecular biology, I have to thank Matt Kiedrowski. Moreover, your friendship has been dear to me in my time at NEOMED. Next, I have to thank Cindy James from Ohio State for all of her help in the optimization of protein mass spectroscopy, while the sample may not have always worked your guidance was appreciated. Finally, I need to send my deepest and upmost gratitude to my advisor Dr. Ian Bratz, he has guided me through the troughs and peaks, the apices and nadirs of science and we have emerged only stronger. We have known each other for over 6 years and grown professionally and personally together. I need to thank you for taking a risk on me as your first graduate student and devoting your time to my development. I thank you for allowing me the freedom to make mistakes. I could always count on you to push me scientifically in our discussions, constantly challenging me, but respecting my opinion concurrently. Thank you Dr. Bratz and thank you everyone. v Vita Biographical Information: Graduate Student, Northeast Ohio Medical University…..….…………….……2012 – 2016 Medical Doctorate Candidate, Northeast Ohio Medical University…………..2010-Present Tutor/Teaching Assistant, Physiological Basis of Medicine, Infection and Immunity…………………………………………………….…………….…….…..2012 – 2014 Publications: 1. Guarini G, Ohanyan VA, Kmetz JG, DelloStritto DJ, Shamhart PE, Thodeti C, Meszaros JG, Damron DS and Bratz IN. Disruption of TRPV1-mediated coupling of coronary blood flow to cardiac metabolism in diabetic mice: Role of nitric oxide and BK channels. Am J Physiol Heart Circ Physiol. May 2012 PMID: 22610171 2. DelloStritto DJ, Connell P, Dick GM, Fancher IS, Klarich B, Fahmy JN, Kang P, Chen Y-R, Damron DS, Thodeti CK, Bratz IN. Differential Regulation of TRPV1 channels by H2O2: Implications for Diabetic Microvascular Dysfunction. Basic Res Cardiol. 2016 Mar;111(2):21 PMID: 26907473 3. DelloStritto DJ, Sinahroy P, Connell P, Fahmy JN, Cappelli H, Thodeti CK, Geldenhuys WJ, Damron DS, and Bratz IN. 4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling. In submission. 4. Doersch KD, DelloStritto DJ, Newell-Rogers MK. The contribution of interleukin-2 to effective wound healing. Experimental Biology and Medicine, In Submission. 5. DelloStritto DJ. Why Societies. Journal of Clinical Investigation, In Progress (To Be Published October 2016). Field of Study: Major Field: Integrated Pharmaceutical Medicine vi Table of Contents Abstract..............................................................................................................................ii Acknowledgements......................................................................................................iii - v Vita....................................................................................................................................vi List of Tables……............................................................................................................viii List of Figures…….......................................................................................................ix - x Chapter 1: Introduction……………………………………...................................................1