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Fibroblast Growth Factor 2-Mediated Cardioprotection: the Kinase Mediators and Downstream Targets of FGF2-Induced Protection from Ischemia and Reperfusion Injury

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Fibroblast growth factor 2-mediated cardioprotection: the kinase mediators and downstream targets of FGF2-induced protection from ischemia and reperfusion injury. A dissertation submitted to the Division of Graduate 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 2012 Janet R. Bodmer Manning BA, Xavier University, 2002 Committee Chair: Jo El J. Schultz ABSTRACT Although heart disease is the primary cause of death in several industrialized nations, there are no widely-used therapies targeting the ischemic heart muscle; current therapies focus on rapid restoration of blood flow, which produces its own set of injuries. Fibroblast growth factor 2 (FGF2) has been shown to protect the heart from ischemia and reperfusion (I/R) injury, reducing infarct size and postischemic dysfunction. However, it is unclear by what mechanism the two classes of FGF2 expressed in the cardiomyocyte, high molecular weight (HMW) FGF2, and low molecular weight (LMW) FGF2, exert protective action on the ischemic heart. It has been established that LMW FGF2 protects the heart from postischemic dysfunction, while endogenously expressed HMW FGF2 reduces contractile function and relaxation after I/R injury. The mechanisms by which this occurs are not well understood. The purpose of this dissertation was to investigate the mechanisms of these differential effects, including elucidating the role of a known cardioprotective kinase, protein kinase C (PKC), in the signal transduction pathways initiated by FGF2 isoforms, as well as the downstream targets of this and other kinases. Of particular interest was determining which isoforms of PKC are mediating LMW FGF2-induced protection from I/R injury, and investigating known and novel targets of these PKC isoforms at the myofibril and sarcoplasmic reticulum that may modulate contractile function. Using mice that only express the LMW FGF2 isoform, it was determined that LMW FGF2 differentially activates PKCε and α, and that these isoforms of PKC were necessary for LMW FGF2-mediated protection. Expression of only LMW FGF2 was also found to increase troponin I and T phosphorylation during ischemia, as well as the activity of actomyosin ATPase, in a manner that was dependent on PKCα. ATPase. Additionally, differences in calcium cycling ii were seen in hearts only expressing LMW FGF2, although no changes in basal levels of calcium cycling proteins were observed. However, a significant elevation of phosphorylated pThr-17 phospholamban was seen at early ischemia, suggesting that this phosphorylation may play a role in LMW FGF2 mediated protection from postischemic dysfunction. Also of interest is the mechanism by which HMW FGF2 reduces postischemic function. It was hypothesized that HMW FGF2 produces its detrimental effects by interfering with protective LMW FGF2 signaling. It was found that after I/R injury, the HMW FGF2 overexpresion results in lowered FGF receptor 1 (FGFR1) activation, which is necessary for LMW FGF2-mediated protection, as well as lowered activity of downstream kinases of FGFR1. Finally, novel targets of both HMW and LMW FGF2 were investigated. It was found using pharmacological methodologies that overexpression of HMW and LMW FGF2 result in a PKC- and MAPK-dependent increase in nitric oxide (NO) production, suggesting that NO synthase (NOS) is a target of FGF2 signaling. In addition, it was found using a microarray that HMW and LMW differentially regulate the expression of genes that may play a potentially protective role in I/R injury. These results elucidate a novel mechanism for a potentially therapeutic molecule to protect the heart from ischemia and reperfusion injury. iii iv ACKNOWLEDGEMENTS This dissertation work would not have been possible without the help of many friends and colleagues, to whom I am greatly indebted. First and foremost, I would like to gratefully acknowledge my advisor and mentor, Dr. Jo El Schultz, for her unwavering support throughout my time as a graduate student. Her careful direction and advice were invaluable to my growth as a scientist, and her method of challenging me to think independently and to defend my research has taught me to become a meticulous, confident experimentalist. I am thankful for her guidance over the years, her advocacy, and her friendship. I would also like to extend my gratitude to the members of my dissertation committee, Drs. W. Keith Jones, Terence Kirley, Evangelia Kranias, Abdul Matlib, and Jeffrey Molkentin, for their encouragement, collaboration, and constructive criticism. The submission of a sound and thorough dissertation project is the result of their meticulous attention to the progress of my research, and my own progress as a graduate student. I am grateful as well to the past and present members of the Schultz laboratory, Gilbert Newman, Dr. Craig Bolte, Adeola Adeyemo, Dan Pietras, Yu Zhang, Angel Whitaker, and Colleen York, for their insights and suggestions, and for joining me in celebration when an experiment worked and in commiseration when it didn’t. I owe a particular debt of gratitude to my student mentor, Dr. Siyun Liao, who spent many patient hours at the bench with me as a new and inexperienced graduate student, and was always available for advice or suggestions, from my first day in the lab to long after her own graduation. I am fortunate to have benefited from the assistance of many collaborators at the University of Cincinnati, including Stela Florea and Wen Zhao in the laboratory of Dr. Kranias, who generously lent their expertise in isolating murine cardiomyocytes and measuring calcium v cycling and cell contraction, and Drs. Aruna Wijeratne and Ken Greis, who put a great deal of hard work into developing a mass spectrometry protocol for determining the relative levels of phosphoproteins. In addition, the collaboration of Dr. W. Glen Pyle and Sarah Parker at the University of Guelph in isolating and analyzing myofilament fractions of the hearts I collected was essential for uncovering the effects of FGF2 isoforms at the myofibril. I would also like to express my gratitude to Dr. Muthu Periasamy and Meghna Pant at the Ohio State University, for their insights and collaboration examining the role of sarcolipin in our heart models. I would also like to thank the medical, graduate, and undergraduate students who have rotated through or volunteered in the Schultz lab, and made significant contributions towards this dissertation, including Brian Oloizia, Greg Carpenter, Laura Moon, Edward Wright, Kristin Luther, Arial Rydeen, Xiaoqian Gao, and Chi Keung Lam. Their enthusiasm brought fresh energy to a variety of projects that were essential for completing the ideas put forth in this manuscript. I owe a tremendous debt of gratitude to the past and current members of the Department of Pharmacology, including Nancy Thyberg, George Sfyris, Carol Ross, Damita Harris, Mark Spanyer, and Donna Gering, for ensuring that deadlines were met, software was working, supplies were ordered, and paperwork was filed. Finally, I would like to thank my friends and family for their love, encouragement, and patience. I offer my gratitude to my parents, for their infectious belief from the very beginning that I could succeed in whatever I wanted to do, and their unconditional support that allowed me to do so under sometimes discouraging circumstances. I would also like to show appreciation for my sister Lisa, for drawing on her own experiences to help talk me through some of the more difficult parts of graduate school, and offering always-welcome advice, and to my brother Bill, vi for helping me to remember to leave the lab occasionally to have fun. I would like to extend my gratitude to my extended family, and in particular my uncle John, for showing a curious fourteen-year-old how a sphygmomanometer worked many years ago. I would also like to thank many of my friends for their patience and encouragement, especially Cate and Solange, who went above and beyond during some of the disheartening moments of the past few years to keep me fed, sheltered, sane, and sufficiently caffeinated to complete my degree. Finally, I’d like to thank my boyfriend Mike, for his unwavering encouragement and love. vii TABLE OF CONTENTS Page Abstract ii Acknowledgments v List of Tables and Figures xiv List of Abbreviations xix Introduction and Background 1 Cardiac Ischemia-Reperfusion Injury 1 Fibroblast Growth Factors and FGF2 5 FGF2 and Cardioprotection 10 High and low molecular weight FGF2 12 Protein Kinase C 15 PKCs and cardioprotection 18 FGF2 and PKCs 20 Downstream targets of FGF2 and PKCs: Contractile proteins 22 Downstream targets of FGF2 and PKCs: Calcium-handling proteins 25 Other targets of FGF2 in the heart during I/R 28 Statement of Purpose 31 Material and Methods 36 Animals and Exclusion Criteria 36 Mouse Generation and Breeding 37 Generation of Fgf2 KO mice 38 viii Generation of high molecular weight FGF2 knockout (HMWKO) mice 39 Generation of low molecular weight FGF2 knockout (LMWKO) mice 42 Generation of PKC alpha KO (PKCαKO) mice 43 Generation of human 24 kDa high molecular weight FGF2 transgenic (HMW Tg) mice 46 Generation of cardiac-specific overexpression of FGF2 transgenic (FGF2 Tg) mice 46 Isolated Working Mouse Heart Ischemia/Reperfusion Studies 49 Time course I/R studies 52 Pharmacological studies 53 Immunoblotting 54 Analysis of FGF2 protein content in heart homogenate 55 Immunoblotting and detection of proteins
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