Environmental Endocrine Disrupting Chemicals and Cardiac Arrhythmogenesis
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Environmental Endocrine Disrupting Chemicals and Cardiac Arrhythmogenesis A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of Pharmacology and Cell Biophysics of the College of Medicine By Xiaoqian Gao B.S. Sichuan University Huaxi Medical Center, 2009 Committee Chairperson: Hong-Sheng Wang, Ph.D. Abstract Environmental endocrine disrupting chemicals (EDCs) are a group of exogenous compounds that may interfere with the functioning of endogenous systems and affect human health. Bisphenol A (BPA) is one of most ubiquitous EDCs in the manufacturing industry as a plasticizing agent used in polycarbonate plastics and epoxy resins. It is well-documented that human exposure to BPA is extremely wide spread. It was demonstrated that BPA, at human-exposure relevant doses, rapidly promoted cardiac arrhythmias in female rat hearts. However, the molecular mechanisms underlying BPA’s pro-arrhythmic effects remain unclear. As a result of banning BPA’s use in various consumer products, bisphenol S (BPS) is increasingly used as a substitute agent for BPA. Human populations are reported to be widely exposed to BPS, but the biological activities and potential toxic effects of BPS are not well understood. The objective of this dissertation is to investigate the cardiac impact of EDCs including BPA and BPS, with a focus on their cardiac arrhythmogenesis and underlying cellular and molecular mechanisms. Of particular interest, was to elucidate the signaling cascades and protein targets underlying BPA’s rapid alteration of myocyte Ca2+ handling and promotion of arrhythmogenic-triggered activities in female rodent hearts; and to evaluate how BPS affects cardiac arrhythmogenesis in comparison to BPA. It was demonstrated that protein kinase A (PKA) and Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathways are the two major signaling pathways activated by BPA. In isolated female rat ventricular myocytes, BPA exposure rapidly increased ii phosphorylation of the ryanodine receptors by PKA but not by CAMKII. BPA exposure also rapidly increased the phosphorylation of phospholamban by CAMKII but not PKA. These two pathways are mediated by estrogen receptor β but not estrogen receptor α, and are shown to be localized. Functional analysis also showed that both PKA and CAMKII were necessary contributors to the arrhythmogenesis of BPA on cardiomyocytes. This study identified the unique signaling cascades of BPA in the heart, and elucidated its novel effects on key Ca2+ handling proteins. Also of interest is the cardiac impact of BPS, especially on the electrical aspect of the heart. It was shown that in female rat hearts, BPS rapidly increased heart rate and promoted ventricular arrhythmias under stress conditions. BPS increased arrhythmogenic-triggered activities in isolated female myocytes via alteration of Ca2+ handling, in particular by increasing spontaneous sarco/endoplasmic reticulum Ca2+ release. BPS exposure increased phosphorylation of two key Ca2+ handling proteins, the ryanodine receptor and phospholamban. Additionally, the pro-arrhythmic effects of BPS were demonstrated to be female-specific, characterized by an inverted-U dose response curve. These results provide important mechanistic insights into the rapid cardiac arrhythmogenesis of BPS in female hearts, and contribute to the evaluation of the potential cardiac toxicity of BPS. Furthermore, the cardiac effects of probenecid were investigated. Collaboratively, it was shown that probenecid increased myocardial contractility using in vivo echocardiography, ex vivo Langendorff perfused heart and isolated myocyte system. The inotropic effect is likely mediated by transient receptor potential vanilloid 2 channels via enhanced sarco/endoplasmic reticulum Ca2+ release. iii iv Acknowledgments First and foremost, I would like to express my deepest gratitude to my thesis advisor, Dr. Hong-Sheng Wang, for being a tremendous mentor for me. Throughout my graduate studies, his careful direction and unwavering support helped me to grow, both academically and personally. His keen sense of science and dedication to research will always influence me in my future career. I am sincerely grateful for his invaluable guidance along this journey. I want to extend my appreciation to the members of my dissertation committee, Drs. Jo El Schultz, Terry Kirley and Steven Kleene, for their insightful advices, constructive criticism and continuous support during my training process. Their constant help with my research and my own progress is indispensible to the completion of this dissertation. My thanks also go to the past and current members of Dr. Hong-Sheng Wang’s laboratory, who are just like my family. I would like to thank Min Dong, Weizhong Song and Sujuan Yan for teaching me key techniques in my projects, Yamei Chen for performing myocyte contractility measurements, Qian Liang and Jianyong Ma for collaborating with me and helping me with troubleshooting, Paul Niklewski for valuable discussions and suggestions. It has been such a pleasure to work with them for all these years, and our friendship will last forever. Special thanks to our collaborator Dr. Jack Rubinstein for his insights and collaborative work in the dissertation. I would like to thank all the faculty and students in the Department of Pharmacology for giving me so much help during my graduate studies. I am truly indebted to Dr. Jo El Schultz, who was our graduate program director, for her constant attention to my study progress and v concern for my personal life. Many thanks to our program coordinator Nancy Thyberg, for always being there to render kind help. I want to thank Janet Manning and Chi Keung Lam for being great teachers for me during my rotations in the first year, and my classmate Clifford Cookman for constant encouragement and support. Finally, my thanks go to my family for their love and continuous support, which have given me strength to overcome the obstacles in both studies and in life. I want to show my appreciation to my parents, for always loving me and believing in me. Especially, I would like to thank my husband Teng, for his unconditional love and being an advocate for every little progress I made. This dissertation would not be completed without his patient help in revision. Lastly, I want to offer gratitude to my baby Aiden, for cheering me up with his beautiful smiles in a tough day, and for helping me to understand the meaning of life. vi Table of Content Page Abstract ..............................................................................................................ii Acknowledgments ............................................................................................v Table of Content .............................................................................................vii List of Abbreviations ........................................................................................xi List of Tables and Figures ...............................................................................xv Chapter I: Introduction .....................................................................................1 1. Environmental endocrine disrupting chemicals ............................................................1 2. Bisphenol A ...................................................................................................................5 3. Bisphenol A and cardiovascular diseases ......................................................................8 4. Cardiac conduction system and ventricular action potential (AP) ...............................10 5. Ventricular arrhythmias and cellular mechanisms .......................................................13 5.1. Premature ventricular beats ................................................................................13 5.2. Ventricular tachycardia ......................................................................................14 5.3. Ventricular fibrillation ........................................................................................16 5.4. Mechanisms of ventricular arrhythmias .............................................................17 5.4.1 Triggered activities: Early after-depolarization and delayed after-depolarization ............................................................................................17 5.4.2 Reentry arrhythmias ..................................................................................18 5.5. Myocyte Ca2+ handling and excitation-contraction coupling .............................20 6. Bisphenol A and cardiac arrhythmias ..........................................................................22 vii 6.1. Experimental evidence .......................................................................................22 6.2. Potential mechanisms ........................................................................................24 7. Bisphenol S ..................................................................................................................27 8. Probenecid ....................................................................................................................29 9. Dissertation focus and hypotheses ...............................................................................31 Chapter II: Materials and Methods ...............................................................35 1. Animals