UNIVERSITY OF CINCINNATI Date: 8-May-2010 I, Michael Wilhide , hereby submit this original work as part of the requirements for the degree of: Master of Science in Molecular, Cellular & Biochemical Pharmacology It is entitled: Student Signature: Michael Wilhide This work and its defense approved by: Committee Chair: Walter Jones, PhD Walter Jones, PhD Mohammed Matlib, PhD Mohammed Matlib, PhD Basilia Zingarelli, MD, PhD Basilia Zingarelli, MD, PhD Jo El Schultz, PhD Jo El Schultz, PhD Muhammad Ashraf, PhD Muhammad Ashraf, PhD 5/8/2010 646 Hsp70.1 contributes to the NF-κΒ paradox after myocardial ischemic insults A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Master of Science (M.S.) in the Department of Pharmacology and Biophysics of the College of Medicine by Michael E. Wilhide B.S. College of Mount St. Joseph 2002 Committee Chair: W. Keith Jones, Ph.D. Abstract One of the leading causes of death globally is cardiovascular disease, with most of these deaths related to myocardial ischemia. Myocardial ischemia and reperfusion causes several biochemical and metabolic changes that result in the activation of transcription factors that are involved in cell survival and cell death. The transcription factor Nuclear Factor-Kappa B (NF-κB) is associated with cardioprotection (e.g. after permanent coronary occlusion, PO) and cell injury (e.g. after ischemia/reperfusion, I/R). However, there is a lack of knowledge regarding how NF- κB mediates cell survival vs. cell death after ischemic insults, preventing the identification of novel therapeutic targets for enhanced cardioprotection and decreased injurious effects. The objective of this thesis is to distinguish and determine the mechanisms underlying the differential effects of NF-κB following ischemic insults. For example, NF-κB-dependent cardioprotection after PO vs. NF-κB-dependent cell death after I/R. It is hypothesized that NF- κB is a key signaling integrator that differentially regulates distinct sets of NF-κB-dependent genes that contribute to cardioprotection or cell death after ischemic insults. Transgenic mice were used, in which NF-κB activation is genetically blocked in the cardiomyocytes (DN), along with gene expression assays (e.g. microarrays and quantitative real-time PCR (QRT-PCR)) to determine sets of genes that may contribute to cardioprotection and cell death. Our results identified 16 genes both up- and down-regulated by NF-κB and after PO, which may contribute to NF-κB-dependent cardioprotection after PO. In addition, our results revealed 59 genes both up- and down-regulated by NF-κB and after I/R, which may result in NF-κB- ii dependent cell death after I/R. The main objective is to identify genes that are dysregulated (up- and down-regulated) between PO and I/R (16 genes regulated by NF-κB and PO vs. 59 genes regulated by NF-κB and I/R). Only one gene was significantly up-regulated by NF-κB after PO and I/R, which is heat shock protein 90kDa alpha (cytosolic), class A member 1 (hsp90aa1). However, heat shock protein 1A (hspa1a/hsp70.3) and heat shock protein 1B (hspa1b/hsp70.1) genes are highly up-regulated in response to both ischemic insults. NF-κB significantly up- regulated hspa1a after PO and hspa1a and hspa1b after I/R. QRT-PCR confirmed that hspa1a and hspa1b genes are highly up-regulated by both ischemic insults and up-regulated by NF-κB. In general, heat shock proteins 70 are known to be involved in cell survival and cell death outcomes. However, there is a lack of information regarding the individual role of Hsp70.1 and Hsp70.3 after myocardial ischemic insults. Therefore, Hsp70.1 and Hsp70.3 were investigated by using Hsp70.1/Hsp70.3 double knockout mice and Hsp70.1 single knockout mice. Interestingly, our results show that Hsp70.1 provided cardioprotection after PO, in contrast to causing cell injury after I/R; whereas, Hsp70.3 may provide cardioprotection after I/R. The significance of the research is the identification of unique sets of genes that may underlie the NF-κB paradox. The novel findings of the thesis are that Hsp70.1 underlies NF-κB-dependent cardioprotection after PO and NF-κB-dependent cell injurious effects after I/R. Our results contribute to the understanding of how NF-κB differentially regulates cell survival versus cell death effects after ischemic stimuli. iii iv Acknowledgments I would like to extend thanks to all the people who provided me with their wonderful advice, support, and encouragement during my graduate career at the University of Cincinnati. First, I would like to thank Dr. W. Keith Jones for the opportunity to do research under his guidance and in his laboratory. I treasure and benefitted from his support, advice, guidance, and encouragement during my graduate career. Dr. Jones is truly a great scientist who is very dedicated to his research and the students in his laboratory. I am very grateful for Dr. Jones’ setting such a high standard for my work and for encouraging me to think critically on research aspects. His commitment of support, mentoring, and advice has guided me through my graduate training and enhanced my professional development. I would also like to extend my appreciation and thanks to the members of my committee: Chairperson Dr. W. Keith Jones, Dr. Muhammad Ashraf, Dr. Mohammad Matlib, Dr. Jo El Schultz, and Dr. Basilia Zingarelli. I am very grateful for their support, encouragement, and dedication to my success as a graduate student throughout my graduate career. I appreciate their guidance in helping me successfully to graduate with a degree. Most importantly, I would like to thank them for their time spent listening and providing advice on my research. My research would not have been possible without the help of Dr. Xiaoping Ren, who gave his time and expertise in the myocardial ischemia and reperfusion surgical models. Without his involvement, my research would not been accomplished. I enjoyed assisting Dr. Ren during the v mice surgeries and talking to him about research. He is truly a wonderful senior member of Dr. Jones’s lab. I am very grateful that I had a chance to work with him and learn from his expertise. I would also like to thank the members of the genomics and microarray laboratory at the University of Cincinnati (UC) for their microarray work. In addition, I extend thanks to the members of the statistical genomics and system biology laboratory at UC, especially Drs. Mario Medvedovic, Maureen Sartor, and Jing Chen who assisted me with microarray analysis. Along with Travis Beckwith, a Ph.D. student in the University of Cincinnati Neuroscience program, who assisted in the editing and proofreading of this thesis. I appreciate the current and past members of Dr. Jones’s lab, who provided their support and skills that contributed to my research. In addition, I would like to extend my gratitude to the following members of the lab: past Ph.D. student Dr. Maria Brown, Michael Tranter (Ph.D. summer 2010) and Jackie Belew (research assistant). Dr. Brown has provided me with wonderful support and advice during my early graduate career, which I appreciate. Michael Tranter provided me with crtical advice and insights on my research. Jackie Belew has provided me with support and encouragement, along with her critical guidance. I am grateful for the faculty and staff of the Department of Pharmacology and Cell Biophysics of the University of Cincinnati for all of their support, advice and encouragement during my graduate career. Special thanks to the following people: Nancy Thyberg, Donna Gering, Carol Ross, Damita Harris, and George Sfryis for their assistance. I would also like to thank the past and current students in the Molecular, Cellular, and Biochemical Pharmacology graduate vi program for their support during my graduate studies. Also, I would like to thank Dr. Carl Huether (a colleague of my mother and UC Professor Emeritus Biological Sciences) for providing me support, advice, and encouragement during my last year of graduate school. I would like to acknowledge the NIH for the predoctoral fellowship and the NIH grants of Dr. W. Keith Jones that provided me with the support and funding for my research. Finally, I am very grateful that God has provided me with a wonderful family who has given me unconditional love and support throughout my life. I am very thankful for all the support that my family (my mother, Margaret Wolf; my step father Bill Wolf; my father, Steve Wilhide; and my brother, Brian Wilhide) has given me, especially during my graduate studies. I would like to dedicate this thesis to my family and in honor of my grandfather, Dr. James D. Weaver who was a wonderful mentor and inspiration to me. vii Table of Contents Abstract ii Acknowledgment v Table of Contents viii List of Figures and Tables xvi Chapter I: Introduction 1 Section 1. General Background 1 I.1.1 Cardiovascular Disease 1 I.1.2 Myocardial Ischemia 1 I.1.3 Ischemic Injury 1 I.1.4 Ischemia/Reperfusion 3 I.1.5 Reperfusion Injury 5 Calcium Paradox 6 Oxygen Paradox 7 I.1.6 Cell Death 8 Apoptosis 9 Table 1. Differences Between Necrosis and Apoptosis 10 Anti/Pro-Apoptotic Genes 12 Extrinsic Death Receptor-Mediated Pathways 12 Intrinsic Mitochondrial-Dependent Pathway 13 Endoplasmic Reticulum (ER)-Stress Cell Death Pathway 14 Figure 1. Cell Death Pathways 16 viii I.1.7 Cell Death After Ischemia and Ischemia/Reperfusion 20 I.1.8 Summary of Background 21 Figure 2. Summary of Background 23 Section 2. Cellular Response to Ischemic and Reperfusion Stresses 24 I.2.1 Unfolded Protein Response 24 I.2.2 Heat Shock Proteins 26 Hsp90 Family 26 Hsp70 Family 27 Hsp60 Family 29 I.2.3 Inflammatory Response 30 I.2.4 Nitric Oxide Synthesis 31 I.2.5 Summary 32 Section 3.