University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 8-2011 Mechanistic insights into copper-induced regression of heart hypertrophy. Katherine Bourcy University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Recommended Citation Bourcy, Katherine, "Mechanistic insights into copper-induced regression of heart hypertrophy." (2011). Electronic Theses and Dissertations. Paper 133. https://doi.org/10.18297/etd/133 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. MECHANISTIC INSIGHTS INTO COPPER-INDUCED REGRESSION OF HEART HYPERTROPHY By Katherine Bourcy B.S., University of Louisville, 2003 M.S., University of Louisville, 2007 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Department of Pharmacology & Toxicology University of Louisville Louisville, KY August 2011 MECHANISTIC INSIGHTS INTO COPPER-INDUCED REGRESSION OF HEART HYPERTROPHY By Katherine Bourcy B.S., University of Louisville, 2002 M.S., University of Louisville, 2008 A Dissertation Approved on July 26, 2011 By the following Dissertation Committee: Y. James Kang, D.V.M., Ph.D. /10hn w. Eator~,iJr,=Ph.D. David W. Hein, Ph.Ir. Zhao-Hui ~ong...Eh.D. Dale.... A. Schuschke, Ph.D. 11 ACKNOWLEDGMENTS I want to thank my dissertation director, Dr. Kang for all his help, guidance and encouragement. I don't know what the future holds, but I have learned so much during my time here and I know that the future is bright. I would also like to extend my gratitude to all my committee members, Drs. Eaton, Hein, Song and Schuschke, for their helpful comments, guidance, their service on my committee, as well as their patience with the last minute scheduling of committee meetings and schedule coordinating messes. I would also like to thank Dr. Cai for his assistance in helping analyze many, many samples using LC/MS-MS, and Dr. Feng for countless hours of assistance and advice, especially while I was trying to get my research up and running and struggling to optimize experimental conditions. I am also very much indebted to Yang Zhou for all her help, especially when I first joined the lab. Additionally, Li Zhan and Lala Hussain have also played an important role in this chapter of my life. And while he was not on my committee, I would also like to thank Dr. Michal Hetman for the life-changing conversation. I would like to thank my friends for their patience and forbearance with my erratic schedule, for their laughter and encouragement that kept me going when all I could think of was how much I wanted to quit. It's been a long time coming, but the tables have finally turned and the day has arrived. I would especially like to thank my sister, Joi. We have been through so much together, from the depths of the pit to the blazing road to the III top that we're on now; I have no doubt I wouldn't have made it without your cheer, wry sense of humor and contagious laughter. I told everyone I wouldjokingly say, "This Ph.D. is brought to you by Sugar-Free Red Bull, sponsored in part by Starbucks Coffee, Heine Brothers, Highland Coffee, Sunergos and Five-Hour Energy!" in my dissertation acknowledgements, but I was actually half serious. In addition to the love and support of all my friends, I would not have survived graduate school without coffee and/or caffeine. Everyone else who has made this journey even the slightest bit more bearable, who was always ready with your quick wit or an encouraging word ... you know who you are, and I love you dearly ... IV ABSTRACT MECHANISTIC INSIGHTS INTO COPPER-INDUCED REGRESSION OF HEART HYPERTROPHY Katherine Bourcy July 26, 2011 Previous studies have shown that copper (Cu) supplementation at physiologically relevant levels reverses cardiac myocyte hypertrophy induced by phenylephrine (PE), and that this effect was VEGF-dependent. Yet, the amount of VEGF in the media was unchanged. However, we observed that Cu caused an increase in the ratio of VEGFR- 1:VEGFR-2 as well as an increase in PKG-1 activity. PKG-1 activity is associated with the regression of cardiac myocyte hypertrophy. The present study was undertaken to test the hypothesis that VEGFR-1 is associated with PKG-1 and their association is involved in Cu induced regression of cardiomyocyte hypertrophy. Human cardiac myocytes (HCM) in cultures were exposed to phenylephrine (PE) at a final concentration of 100 flM for 48 hours to induce cell hypertrophy. Copper sulfate at a final concentration of 5 flM was added to the hypertrophic HCM cultures for 24 hours with the concomitant presence of PE to reverse the hypertrophy. Both hypertrophic and hypertrophic-reversed HCM cells underwent immunoprecipitation using anti-VEGFR-1 antibody or anti-PKG-1 antibody. The immune complex underwent gel-electrophoresis separation and Western blotting and LC-MSIMS analysis. Proteomic analysis identified Vimentin in the immune v complexes that immunoprecipitated with VEGFR-l and PKG-l. This study thus demonstrates that the association between VEGFR-l with PKG-l is mediated by Vimentin, and that Vimentin plays a critical role in copper regression of cardiac myocyte hypertrophy. vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS .......................................................................... .iii ABSTRACT ........................................................................................... v LIST OF TABLES ................................................................................. viii LIST OF FIGURES .................................................................................. .ix I. Introduction ................................................................................... 1 1.1 The Heart & Heart Failure ....................................................................... 5 1.2 Element #29: Cuprum ......................................................................... .1 0 1.3 Vascular Endothelial Grown Factor and its Receptors .................................... .16 1.4 PKG-1 ....... , .................................................................................... 22 1.5 Vimentin ............................................................................................... 26 1.6 Hypothesis ....................................................................................... 30 II. Materials and Methods .......................................................................... 31 III. Results ...............................................................................................49 IV. Discussion ....................................................................................... 126 REFERENCES ..................................................................................... 133 APPENDIX .......................................................................................... 150 CURRICULUM VITAE .......................................................................... 153 Vll LIST OF TABLES Page Table 1: PKG-l pull down ....................................... , .............................. 85 Table 2: PKG-l pull down ......................................................................96 Table 3: PKG-l pull down .................................................................. 107 Table 4: VEGFR-l pull down ............................................................... 112 Vlll LIST OF FIGURES FIGURE Page 1. Quantitative analysis of VEGF secretion .................................................50 2. Schematic representation of VEGFR signaling .......................................... 51 3. Fluorescent microscopic images ofVEGFR-l and VEGFR-2 ........................ 53 4. Flow cytometric analysis of VEGFR-l and VEGFR-2 ................................ .54 5. Flow cytometric analysis ofVEGFR-l and VEGFR-2 ................................. 55 6. Quantitative analysis of VEGFR-l density ............................................... 57 7. Quantitative analysis of VEGFR-2 density ............................................... 58 8. Regression of PE-induced cell hypertrophyby siRNA targeting VEGFR-2 ......... 59 9. Regression of PE-induced cell hypertrophy by siRNA targeting VEGFR-2 ......... 60 10. Regression of PE-induced cell hypertrophy by siRNA targeting VEGFR-2 .......61 11. Requirement ofVEGF .....................................................................63 12. Requirement of VEGF ..................................................................... 64 13.Requirement of VEGF ..................................................................... 65 14. Requirement of VEGF ..................................................................... 66 15. Effect of siRNA targeting VEGFR-1. .................................................... 68 16. Effect of siRNA ............................................................................ 69 17. Quantitative analysis ofVEGFR-l post-siRNA targeting VEGFR-1. .............. 70 18. Quantitative analsys of cell size post-siRNA targeting VEGFR-l .................. 71