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A Dissertation Entitled Regulation of Na/K-Atpase and Its Role In A Dissertation entitled Regulation of Na/K-ATPase and its Role in Cardiac Disease by Xiaoming Fan Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences ________________________________________ Dr. Jiang Tian, Committee Chair ________________________________________ Dr. Andrew D. Beavis, Committee Member ________________________________________ Dr. Kevin Z. Pan, Committee Member ________________________________________ Dr. David Giovannucci, Committee Member ________________________________________ Dr. Lijun Liu, Committee Member ________________________________________ Dr. Christopher J. Cooper, Committee Member ________________________________________ Dr. Cyndee Gruden, Dean College of Graduate Studies The University of Toledo December 2018 Copyright 2018, Xiaoming Fan This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Regulation of Na/K-ATPase and its Role in Cardiac Disease by Xiaoming Fan Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo December 2018 Heart failure is an important public health issue and a leading cause of mortality in the United States. Previous publications have shown that both protein amount and enzyme activity of cardiac Na/K-ATPase were reduced in heart failure patients. Analysis of gene expression database also demonstrated that expression of Na/K-ATPase α1 subunit in heart tissue from heart failure patients is lower compared to non-heart failure patients. However, it is not clear whether Na/K-ATPase reduction is causatively related with heart failure, and if so, how Na/K-ATPase expression is regulated. During the past twenty years tremendous work have been done to show that Na/K-ATPase, especially its signaling function, is associated with cardiac hypertrophy and cardiac fibrosis. Interestingly, data from our laboratory showed that reduction of Na/K-ATPase attenuates its signaling activation, which might be beneficial to reducing cardiac hypertrophy, but it also increases cardiac cell apoptosis. The aim of my project is to: 1) study how Na/K-ATPase is regulated during disease conditions; and 2) study the effect of Na/K-ATPase reduction on cardiac function and its mechanism. From these studies, we have identified a novel long-non-coding RNA, ATP1A1-AS1, as an endogenous Na/K-ATPase regulator that affect Na/K-ATPase expression and its signaling function (Chapter 3). We also demonstrated that reduction of iii Na/K-ATPase α1 had significant effect on cardiac function and remodeling due to the change in Na/K-ATPase signaling (Chapter 4). ATP1A1-AS1 gene is a natural antisense gene of Na/K-ATPase α1 (ATP1A1) which is located on the opposite strand of the Na/K-ATPase α1 gene. Our results showed that 4 splice variants expressed in human adult kidney cells (HK2 cells) and embryonic kidney cells (HEK293 cells). We found that inhibition of DNA methylation had a differential effect on the expression of ATP1A1-AS1 and its sense gene. To investigate the physiological role of this antisense gene, overexpression of ATP1A1-AS1 was performed and its effect on Na/K-ATPase expression was examined. The result showed that ATP1A1- AS1-203 overexpression reduced the Na/K-ATPase α1 (ATP1A1) gene expression in HK2 cells by about 20% (p<0.05), it also reduced the Na/K-ATPase α1 protein by about 22% (p<0.05). In addition, overexpression of ATP1A1-AS1-203 attenuated ouabain-induced Src activation in HK2 cells and subsequently inhibited the cell proliferation in the presence or absence of ouabain. These results demonstrate that ATP1A1-AS1 gene is a moderate negative regulator of Na/K-ATPase α1 and can modulate Na/K-ATPase-related signaling pathways. Importantly, these results suggest that a moderate reduction of Na/K-ATPase expression could disproportionally affect the signaling function of Na/K-ATPase, which is consistent to the previous findings in Na/K-ATPase knockdown cell lines. To study the effect of Na/K-ATPase on cardiac function in disease conditions, we used a mouse chronic kidney disease (CKD) model (5/6th partial nephrectomy or PNx) in Na/K-ATPase alpha 1 heterozygous (α1+/-) mice and their wild type (WT) littermates. The cardiac Na/K-ATPase alpha 1 expression in α1+/- mice is about 40% less than that of WT mice. The experimental results showed that reduction of Na/K-ATPase significantly iv reduced cardiac hypertrophy in the CKD animals. However, it showed no significant change in cardiac fibrosis between the two animal strains. To further study the role of Na/K-ATPase in cardiac fibrosis, we found that CKD induces activation of Src and NFB signaling in the heart tissue of WT mice, which subsequently causes reduction of microRNA-29b-3p (miR-29b), an antifibrotic microRNA. However, in α1+/- mice, Src and NFB activation was significantly attenuated. We further found that in WT mice, inhibition of Src signaling using pNaKtide blocked NFB activation and restored miR-29b expression to a level close to the controls. Whereas, the pNaKtide had no significant effect on Src/NFB activation or miR-29b expression in α1+/- mice. Injection of pNaKtide also significantly reduces cardiac fibrosis in WT mice. These results suggest that Na/K-ATPase reduction not only attenuates Na/K-ATPase signaling function, it may also adopt other pathological pathways, which can cause cardiac fibrosis independent of Na/K-ATPase- related Src and NFB signaling. The specific mechanisms are elusive and merit further investigations. v To my wife, Dr. Xiaolu Zhang, for your love and support during my Ph.D. study. To my parents, Hansong Fan and Xuanfen Luo, for your love, support, and encouragement throughout my life. To my son, Theo Fan, for the joy you brought to my life. vi Acknowledgements I am extremely grateful to Dr. Jiang Tian for his guidance and faithful support throughout my graduate studies in both academic and personal life. I am looking forward to work with you in the future. I would like to thank my academic advisory committee Dr. Andrew Beavis, Dr. Kevin Z. Pan, Dr. David Giovannucci, Dr. Christopher J. Cooper and Dr. Lijun Liu, for their expert advice and guidance throughout my graduate studies. I would also like to appreciate the support from the Department of medicine, especially the support from Dr. Lance Dworkin. My thanks also go to the people in Dr. Kumarasamy’s lab, Dr. Kennedy’s lab, Dr. Haller’s lab, Dr. Gupta’s lab, and Dr. Gong’ lab for the great collaboration and help during my graduate study. A special thanks to Dr. Shihe Liu for his friendly help. I would especially like to thank all my former and current colleagues Dr. Christopher Drummond, Dr. Huilin Shi, Xie, Dr. Jeffrey Xinshuo Xie and Mano Tillekeratne, for all the great discussion, inspiration and friendship. vii Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ........................................................................................................... vii Table of Contents ............................................................................................................. viii List of Tables .......................................................................................................................x List of Figures .................................................................................................................... xi List of Abbreviations ....................................................................................................... xiii 1. Introduction ......................................................................................................................1 2. Literature review ..............................................................................................................4 2.1 Na/K-ATPase biology ........................................................................................ 4 2.1.1 Structure of Na/K-ATPase ..................................................................... 4 2.1.2 Ion pumping function of Na/K-ATPase ................................................. 6 2.1.3 Ligands of Na/K-ATPase ....................................................................... 7 2.1.4 Signaling function of Na/K-ATPase ...................................................... 8 2.2 Heart failure and cardiac remodeling ................................................................. 9 2.3 Na/K-ATPase and cardiac remodeling ............................................................. 11 2.4 Regulation of Na/K-ATPase ............................................................................ 14 3. Characterization of A Long Non-Coding RNA, The Antisense RNA of Na/K-ATPase Alpha 1 In Human Kidney Cells ........................................................................................16 viii Abstract .................................................................................................................. 17 Introduction ............................................................................................................ 18 Results .................................................................................................................... 20 Discussion .............................................................................................................
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