The Role of Tsg101 in the Development of Physiological Cardiac Hypertrophy and Cardio-Protection from Endotoxin-Induced Cardiac Dysfunction A dissertation to be submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Pharmacology and Systems Physiology, College of Medicine By: Kobina Q. Essandoh B.A. in Biochemistry from Cornell College, 2011 Advisor and Committee Chair: Guo-Chang Fan, Ph.D. Abstract In this dissertation, the functional role of Tumor susceptibility gene (Tsg101) in the regulation of physiological cardiac hypertrophy and endotoxin-induced cardiac dysfunction was explored. Development of physiological cardiac hypertrophy has primarily been ascribed to the insulin-like growth factor 1 (IGF-1) and its receptor, IGF-1R, and subsequent activation of the Akt pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiological hypertrophy has not been investigated. Furthermore, cardiac mitochondrial damage and subsequent inflammation are hallmarks of endotoxin-induced myocardial depression. Activation of the Parkin/PINK1 pathway has been shown to promote autophagy of damaged mitochondria (mitophagy) and protect from endotoxin-induced cardiac dysfunction. Tsg101 has been demonstrated to play diverse roles in the cell including virus budding, cytokinesis, transcriptional regulation, endosomal recycling of receptors and activation of autophagic flux. Hence, the first goal of this dissertation was to elucidate the role of Tg101 in endosome-mediated recycling of IGF-1R in physiological cardiac remodeling. The second goal of this dissertation was to investigate whether Tsg101 regulates mitophagy and thus contribute to endotoxin-caused myocardial dysfunction. Firstly, in a physiological hypertrophy model of treadmill-exercised mice, we observed that levels of Tsg101 were dramatically elevated in the heart, compared to sedentary controls. To determine the role of Tsg101 on physiological hypertrophy, we generated a transgenic mouse model with cardiac-specific overexpression of Tsg101. These transgenic (TG) mice exhibited physiological cardiac hypertrophy at 8 weeks, evidenced by significant enhancement of cardiac function without fibrosis, increased total and membrane levels of IGF-1R, as well as Akt activation, compared to wild-types. Mechanistically, we identified that Tsg101 interacted with i FIP3 and IGF-1R, thereby stabilizing the endosomal recycling compartment (ERC) and enhancing recycling of IGF-1R. In vitro, adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of 1) Monensin, an inhibitor of endosomal recycling; 2) Picropodophyllin, an inhibitor of IGF-1R signaling; and 3) siRNA-FIP3. Furthermore, knockdown of Tsg101 in both mice and neonatal cardiomyocytes significantly inhibited the expression of Rab11a and FIP3 and endosomal recycling of IGF-1R, compared to controls. Interestingly, inducible Tsg101-knockdown mice failed to develop cardiac hypertrophy after treadmill training. Additionally, Tsg101-TG were protected from cardiac fibrosis and dysfunction associated with pathological hypertrophy, induced by transverse aortic constriction surgery. Secondly, Tsg101-TG and -KD mice underwent endotoxin (LPS) treatment (10μg/g) to determine survival, cardiac function, systemic/local inflammation, and activity of mitophagy mediators in the heart. Upon endotoxin challenge, Tsg101-TG mice exhibited decreased mortality, preserved cardiac contractile function, reduced inflammation, enhanced activation of mitophagy in the heart and preservation of mitochondrial structural integrity, compared to control mice. By contrast, endoxin treatment in Tsg101-KD mice exacerbated animal mortality, cardiac dysfunction, inflammation and mitochondrial structural damage. Both co-immunoprecipitation assays and co-immunofluorescence staining showed that Tsg101 was bound to Parkin in the cytosol of myocytes and consequently facilitated translocation of Parkin to the mitochondria. Altogether, this dissertation demonstrates that Tsg101: a) regulates physiological cardiac hypertrophy through the FIP3-mediated endosomal recycling of IGF-1R; and b) could protect against endotoxin-triggered myocardial injury by promoting Parkin-induced mitophagy. ii iii Acknowledgements First and foremost, I am extremely grateful to my thesis advisor, Dr. Guo-Chang Fan for being a great mentor during my time in his lab. His guidance and mentorship has provided me with a strong foundation as a scientist. His encouragement in times of difficulty and his enthusiasm when goals and achievements are attained have enabled me in this journey. His dedication to science has had a big effect on my personal and professional development and these values will key as I develop in my research career. I am very appreciative to my thesis committee members, Drs. Evangelia Kranias, Terry Kirley, Jack Rubinstein and Charles Caldwell, for the suggestions and criticisms that they provided me in our meetings. The insightful comments and advice have nurtured my critical thinking skills which have been instrumental to the completion of this thesis. Their suggestions and help have shaped my dissertation to the form that it is and I am blessed to have them on my dissertation committee. I am very thankful to past and present members of the Fan laboratory, Drs. Liwang Yang, Dongze Qin, Jiangtong Peng, Haitao Gu and Xingjiang Mu for their friendship, encouragement and input into my project. I am especially indebted to Dr. Xiaohong Hong for her mentorship and for teaching me various techniques early on when I joined the Fan lab. I am also very appreciative of Dr. Shan Deng, who dedicated her time and efforts in helping me with experiments. I would like to show my gratitude to the collaborators whose work enhanced the quality of this dissertation. I would like to thank Ming Jiang and Nathan Robbins of the Rubinstein lab for the hours spent in performing and analyzing echocardiographic data. I am also thankful to the lab of Dr. Yigang Wang for providing and allowing me to utilize their equipment. Especially, I would iv like to thank Wei Huang, for assisting me with echocardiography and surgical procedures. I would like to extend my appreciation to Dr. Kay-Uwe Wagner for providing our lab with the Tsg101 floxed mice, which has enabled me to complete this thesis. I am very lucky to have encountered great individuals in Department of Pharmacology and Systems Physiology. I would like to show my gratitude to Drs. Abdul Matlib, Robert Rapoport and John Maggio for their mentorship and advice during my time in both the Masters’ and Doctoral programs. I would like to appreciate Nancy Thyberg for her kindness, support and for always having an open door in times that I needed help. I am thankful to my classmates and fellow graduate students, Yutian Li, Fawzi Alogaili, George Gardner, Shaimaa Ibrahim for their collaboration and friendship through out this journey. I am honored to be have been awarded two fellowships during my time in graduate school. I was honored to be awarded the Albert J. Ryan fellowship that provided support for my research and gave me an opportunity to network with other fellows at our annual meeting. I am also thankful for the American Heart Association Pre-doctoral fellowship which provided stipend support. Lastly, I would like to thank my family for the support they have provided me over the years. I would like to thank my parents, Akwasi and Alberta, for the sacrifices they made for me to pursue an education away from home. I owe a tremendous amount of gratitude to them for their patience, love, encouragement and always being there for me. I am very appreciative of my siblings, Kofi, Jackie and Abeiku, for being my biggest cheerleaders and encouraging me throughout my studies. v Table of Contents Abstract i Acknowledgements iv Table of Contents 1 List of Abbreviations 7 List of Figures and Tables 13 Chapter I Introduction 17 Section 1 Role of IGF-1R in Physiological Hypertrophy 17 I.1.A Overview of Cardiac Hypertrophy 17 I.1.B Characteristics of physiological versus pathological cardiac hypertrophy 18 I.1.C Molecular mechanisms involved in cardiac hypertrophy 20 I.1.D IGF-1R/Akt signaling and physiological cardiac hypertrophy 21 I.1.E IGF-1R and endosomal system 23 Section 2 Mitochondrial dysfunction in Septic cardiomyopathy 26 I.2.A Overview of Sepsis 26 I.2.B Septic cardiomyopathy 30 I.2.C Mitochondria dysfunction in septic cardiomyopathy 31 I.2.D Mitophagy/Autophagy in septic cardiomyopathy 34 Section 3 Tumor Susceptibility Gene 101 (Tsg101) 35 I.3.A Discovery, Structure and Expression of Tsg101 35 I.3.B Role of Tsg101 in cancer 37 I.3.C Role of Tsg101 in cell proliferation 39 I.3.D Role of Tsg101 in the ESCRT machinery 40 I.3.E Role of Tsg101 in HIV budding 41 1 I.3.F Role of Tsg101 in receptor recycling 42 I.3.G Role of Tsg101 in autophagy 43 Section 4 Dissertation Scope and Objectives 44 Chapter II Materials and Methods 47 Section 1 Generation of Mouse Models 47 II.1.A Generation of Tsg101 Transgenic Mice 47 II.1.B Generation of Tsg101-Knockdown Mice 49 Section 2. Exercise-Induced Cardiac Hypertrophy model of Treadmill Training 51 Section 3. Mouse model of endotoxemia 51 Section 4. Determination cardiac contractile function 52 II.4.A In vivo Measurement of cardiac function 52 II.4.B In vitro cardiomyocyte isolation and Measurement of Mechanics 52 Section