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View Highlighting the Importance of a Flexible and Multidisciplinary Approach,” J INVESTIGATING CARDIAC METABOLISM IN BARTH SYNDROME USING INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES ERICA M. FATICA Bachelor of Science in Pharmaceutical Science Cleveland State University May 2014 Submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL-BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY May 2019 We hereby approve this dissertation For Erica Marie Fatica Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for the Department of Chemistry And CLEVELAND STATE UNIVERSITY’S College of Graduate Studies by Committee Chairperson, Yana Sandlers Department of Chemistry, April 18th, 2019 Committee Member, Dr. David Anderson Department of Chemistry, April 18th, 2019 Committee Member, Dr. Michael Kalafatis Department of Chemistry, April 18th, 2019 Committee Member, Dr. Christine Moravec Department of Biological, Geological and Environmental Sciences, April 18th, 2019 Committee Member, Dr. Aimin Zhou Department of Chemistry, April 18th, 2019 April 18th, 2019 Date of Defense Dedicated to my parents/biggest fans In loving memory of Angela Grandillo, whose dedication to never stop learning will forever inspire me ACKNOWLEDGEMENTS This degree would not have been possible without the amazing people who have supported me along the way. First, I would like to sincerely thank my advisor, Dr. Yana Sandlers, for taking me into her research group and for being the most supportive, understanding, and wonderful mentor I could have ever asked for. Thank you for all the opportunities you gave me which allowed me to grow as a scientist and as an individual. Thank you for always believing in me and for encouraging me to have more confidence in myself. I would not have been able to go this far without you. I would also like to thank my truly amazing dissertation committee members. Without Dr. David Anderson’s guidance during my undergraduate work, I would not have stayed at Cleveland State to continue my studies. Thank you for your continued support, care, and guidance in all things clinical chemistry. I am extremely grateful to have had such a wonderful and encouraging female role model in Dr. Christine Moravec. I am thankful to Dr. Aimin Zhou, who has always been the most kind person you could hope to know. Thank you to Dr. Michael Kalafatis for always having my back and supporting me. A special thank you to Dr. Vania DePaoli for always being there for me in every aspect of my life and for looking out for me. I will miss our chats in your office. Also, thanks to Dr. DiBello for being so sweet and supportive, encouraging and inspiring. You are a truly great educator and person, and I hope to carry those qualities with me. I also would like to acknowledge my labmates, especially Rohan Shah for being with me every step of the way (and buying us cookies) and Igor Radzikh, for being my right-hand man. Igor, without you, many things would not have been possible. Thank you also to Ryan, Jared, and Jill for helping me along the way. Many thanks to SooYeon Kang, for all her hard work and assistance. Thank you to my mom, Elia Iafelice, who literally kept me alive during these five years. Without you, I probably would not have eaten for five years. Thank you for putting up with me and for reminding me to take care of myself sometimes. Thank you to my dad, Ron, for always being interested in my work and for helping me troubleshoot technological things. Thank you to my brother, Marco, for helping me understand how to do some of the complicated math stuff that I used for my research project. Your and dad’s ability to understand and work with completely new information never ceases to amaze me. Also thank you to Liz Grandillo, the woman who looks like she could be my mom and acts like my sister, gives me cute hand-me down clothes, lets me visit her in New York to raid her closet, and does not understand anything about cardiac function, except for having occasional chest pains (and thanks for dictating your own acknowledgement). To my best friend, Alana, and our daily chats. Thanks for escorting me in and out of school with your ridiculous stories. Thank you, Alwilleed, for supporting me in every way, reminding me to stay tough, and for bringing me so much joy. To everyone else who helped me over the past five years, especially my classmates, your moral support and love helped carry me through. Your presence in my life made a difference and I am truly grateful. INVESTIGATING CARDIAC METABOLISM IN BARTH SYNDROME USING INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES ERICA M. FATICA ABSTRACT Barth syndrome (BTHS) is an X-linked genetic disorder characterized by cardiomyopathy, neutropenia, skeletal muscle weakness, and 3-methylglutaconic aciduria (Sandlers, et al., 2016). The mortality rate of BTHS patients is high during infancy and childhood due to sudden cardiac death. Despite the severity of this disease, there is a lack of targeted therapeutics which can be used to ameliorate symptoms and prolong the lives of BTHS patients. A major obstacle for the discovery of new therapeutic targets is poor understanding of the mechanisms of cardiac pathogenesis and downstream metabolic effects. To overcome this barrier, we developed a model of BTHS, using human-induced pluripotent stem cells (iPSCs), with or without the BTHS-causative TAZ mutation, to produce functional cardiomyocytes (iPS-CMs). iPS-CMs recapitulate the human donor genotype, reproducing the complex metabolic conditions of the hearts of affected individuals, and permitting investigation of molecular and metabolic mechanisms. We further applied stable isotope-labeled energy substrates and mass spectrometric analyses to our TAZ-mutant iPS-CM model to dynamically trace the fates of substrates through metabolic pathways, including fatty acid oxidation, glucose oxidation, and select anaplerotic pathways into the citric acid cycle (CAC). The studies herein provided novel insight into downstream metabolic differences in energy substrate metabolism between vi control and TAZ-mutant iPS-CMs, revealing alterations in several pathways which can be further explored to uncover potential therapeutic targets for BTHS patients. vii TABLE OF CONTENTS Page ABSTRACT………………………………………………………………………...……vi LIST OF FIGURES……………………………………………………………………. xxi CHAPTER I. INTRODUCTION……………………………………………………………... 1 1.1 Barth Syndrome……………………………………………..…..…… 1 1.2 Tafazzin and Cardiolipin………………………………….................. 2 1.3 Current Treatment Strategies for Barth Syndrome Patients………..... 4 1.4 Dilated Cardiomyopathy………………………………….……...…... 6 1.5 Cardiac Metabolism…………………………………………...….….. 6 1.6 Cardiac Metabolism in Pathophysiological Conditions……...…….… 9 1.7 Cardiac Anaplerosis……………………………………...……….… 12 1.8 Therapeutic Strategies for Modulating Cardiac Metabolism…...…... 15 1.9 Induced Pluripotent Stem Cells………………………..………….... 16 1.10 Stable Isotope Metabolomics………………………...……………. 19 1.11 References………………………………………...……………….. 22 II. ESTABLISHING A MODEL OF BARTH SYNDROME USING INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES…..……. 29 2.1 Abstract…………………………………………...…………….…... 29 2.2 Introduction…………………………………...…………….………. 30 2.3 Methods…………………………………...………………….…...… 35 2.4 Results………………………………...……….……….…................ 39 2.5 Discussion……………………………...……………………….…... 44 viii 2.6 References…………………………..…………………………....… 51 III. STABLE ISOTOPE TRACING STUDY IN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES TO INVESTIGATE METABOLIC ALTERATIONS IN BARTH SYNDROME………………. 51 3.1 Abstract…………………………………………...…………….…... 51 3.2 Introduction…………………………………..……….……………. 52 3.3 Methods…………………………………..……………………....… 54 3.4 Results………………………………..……………….…................. 60 3.5 Discussion………………………...……………………………….... 72 3.6 References……………………...………………………................… 77 IV. ANAPLEROTIC PATHWAYS OF THE CITRIC ACID CYCLE IN TAZ- MUTANT INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES…………..……………………………………...…. 81 4.1 Abstract…………………..………………………….……………... 81 4.2 Introduction…………..……………………………………….……. 82 4.3 Materials and Methods…………..………….…………...…………. 86 4.4 Results………………………..……………………….…................. 89 4.5 Discussion………………..………………………………….……... 99 4.6 References…………..………………………………….……….… 105 V. CALCIUM CHANNEL EXPRESSION IN TAZ-MUTANT INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES..…….. 109 5.1 Abstract………………………………………..………….………. 109 5.2 Introduction to Calcium Channel Proteins…………..….……….... 110 ix 5.3 Materials and Methods………………………………………….… 114 5.4 Results……………………………………..………….…………... 116 5.5 Discussion…………………………..…………………………….. 122 5.6 References……………………..………………………………..… 128 VI. GENERATION OF INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES IN 3D ON A 384-MICROPILLAR PLATE……... 133 6.1 Abstract………………………………..…...…………...…………. 133 6.2 Introduction……………………………....………………………... 134 6.3 Methods…………………………......……...………….…………... 138 6.4 Results………………………………………….…….……………. 143 6.5 Discussion……………………………………….………………… 155 6.6 References………………………………….………….…………... 161 VII. CONCLUSION ……...…………………………………………............... 164 7.1 References…………………………...………………….…………. 166 APPENDICES A. CORRECTION OF MASS ISOTOPOMER DISTRIBUTIONS FOR NATURAL ISOTOPIC ABUNDANCE……………..………………….... 169 B. GAS CHROMATOGRAPHY-MASS SPECTROMETRY METHODS…. 177 x LIST OF FIGURES Figure Page 1.1 Cardiac Energy Substrates ......................................................................................... 8 1.2 ATP Yield and Oxygen Consumption. ...................................................................
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