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A Study of the SERCA-Phospholamban Regulatory Interaction Using Time-Resolved Fluorescence

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A Study of the SERCA-Phospholamban Regulatory Interaction Using Time-Resolved Fluorescence A Study of the SERCA-Phospholamban Regulatory Interaction Using Time-Resolved Fluorescence A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Daniel Richard Stroik IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY David D. Thomas, Advisor August 2018 Daniel Richard Stroik 2018 © Acknowledgements It is a difficult task to thank everyone in a laboratory as expansive and layered as the DDT group; but I will begin by thanking my advisor, Dr. David Thomas. I always appreciated his willingness to help others without prejudice or profit, and it is no surprise that this has afforded him many friends and allies over the years. He has always pushed me and for this I am grateful. My time here has made me excited for a future career in science and ready to go after any discovery. I would like to thank my committee Dr. John Lipscomb, Dr. Wendy Gordon, and Dr. Sivaraj Sivaramakrishnan. They have provided me with guidance and support, and I have always found useful knowledge and inspiration through our interactions. Because of the directions that my thesis project took, I worked very closely with Dr. Razvan Cornea and Dr. Mike Autry. I have always enjoyed their feedback and am very lucky to be surrounded by positive role models. I aspire to be as strong a writer and as clear-thinking a scientist as Razvan. Mike has always been supportive and is one of the most knowledgeable scientists I have ever met. The time he takes to train his students impresses me and he is truly a man of integrity. Many thanks to the lab managers Sarah Blakely, Octavian Cornea and Destiny Ziebol for their assistance in all matters. There is no doubt that the lab would collapse without you. To Dr. Robyn Rebbeck and Samantha Yuen, without a doubt the lab would collapse and/or be ablaze if not for your presence as well. Thanks for the conversations and friendship! I have always been impressed by your work ethic. All the extra deeds (e.g., cakes, Town Hall meetings, get-well cards, farewell parties) make the DDT lab a very positive environment to work in. Thanks to Dr. Simon Gruber for taking the time to train me during my rotation. Lab members Dr. Tory Schaaf, Peter Martin, Yahor Savich, Mark i Rustad, and Evan Kleinbohl have made the overall experience interesting and enjoyable, and I am grateful for that. I have had many interesting and impactful conversations with Dr. Ben Binder, Mike Fealey, and Prachi Bawaskar. Three undergraduates, Skylar Faussner, Kevyn Janicek, and Paul Thanel, have assisted me over the years, and each showed a dedication to lab that is inspirational. I have always been passionate about teaching and they gave me a chance to put the passion into practice. I wish them the best in their future endeavors! ii Dedication To Susanna Jane Huggenberger iii Abstract Cardiac muscle contraction and relaxation is controlled by changes in intracellular Ca, indicating that Ca transport is a fundamental regulator of proper muscle function in the heart. The primary cardiac Ca transporter is the sarcoendoplasmic reticulum Ca- ATPase 2a (SERCA2a), a transmembrane protein pump embedded in the sarcoplasmic reticulum (SR). SERCA2a translocates two Ca ions from the cytosol into the SR at the cost of one ATP molecule, effectively lowering the cytosolic Ca concentration and inducing cardiomyocyte relaxation. Ca transport is regulated by a second transmembrane protein, phospholamban (PLB), which binds to SERCA2a and inhibits its activity by reducing the Ca affinity of the Ca pump. The significance of PLB-dependent regulation in muscle function is highlighted by the existence of hereditary mutations in PLB linked to cardiomyopathy. Further, the membrane protein complex between SERCA2a and PLB is a validated therapeutic target for reversing cardiac contractile dysfunction in more common diseases (e.g., heart failure) caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. The work presented in this thesis focuses on the structure and function of the SERCA2a-PLB complex and its connection to several chronic cardiac diseases. In the first study (Chapter 4), we developed a structure-based high-throughput screening (HTS) method to discover compounds that disrupt the SERCA2a-PLB interaction. We identified ten compounds that reproducibly alter SERCA2a-PLB structure and function, including one compound that increases SERCA2a calcium affinity in cardiac SR membranes but not in skeletal. These results suggest that the compound is acting specifically on SERCA2a- PLB, as needed for a drug to mitigate deficient Ca transport in heart failure. In the second study (Chapter 5), we studied the effects of the disease-causing R9C-PLB mutation in a iv human induced pluripotent stem cell (hiPSC) line to understand the mechanistic details of hereditary cardiomyopathy progression. In addition to blunted lusitropic response to β- adrenergic stimulation, we found that the R9C mutation results in an altered metabolic state and significant gene expression changes. Ongoing studies (Chapter 6) are presented, focusing on recent work to develop PLB-based gene therapy constructs and elucidate the structural mechanism by which a third transmembrane protein, dwarf opening reading frame (DWORF), regulates Ca transport in the heart. v Contents List of Figures ................................................................................................................ vii List of Equations ........................................................................................................... viii List of Tables .................................................................................................................. ix Chapter 1 – Calcium Signaling in Cardiac Muscle .......................................................... 1 1.1 The Role of Calcium in Excitation-Contraction Coupling ................................... 1 1.2 The SR Ca-ATPase .......................................................................................... 4 1.3 Phospholamban and DWORF .......................................................................... 8 1.4 SERCA-PLB and SERCA-DWORF Regulatory Models ...................................12 Chapter 2 – SR Ca-ATPase Activity in Healthy and Diseased States ............................16 2.1 Altered SR Ca Cycling in Cardiac Disease ......................................................16 2.2 Therapeutic Approaches for Heart Failure .......................................................17 2.3 Inherited Cardiomyopathies Caused by PLB Mutations ...................................24 Chapter 3 – Principles of Fluorescence .........................................................................25 3.1 Phenomena of Fluorescence and Phosphorescence .......................................25 3.2 Fluorescence Resonance Energy Transfer......................................................27 3.3 Steady-State and Time-Resolved Fluorescence ..............................................30 Chapter 4 – Targeting Protein-Protein Interactions for Therapeutic Discovery via FRET- Based High-Throughput Screening in Living Cells .........................................................34 4.1 Outline .............................................................................................................35 4.2 Introduction .....................................................................................................35 4.3 Methods ..........................................................................................................39 4.4 Results ............................................................................................................46 4.5 Discussion .......................................................................................................55 4.6 Supplementary Information .............................................................................61 Chapter 5 – Functional and Transcriptomic Insights into Pathogenesis of R9C Phospholamban Mutation using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes .............................................................................................................70 5.1 Outline .............................................................................................................71 5.2 Introduction .....................................................................................................71 5.3 Methods ..........................................................................................................74 5.4 Results ............................................................................................................76 5.5 Discussion .......................................................................................................86 5.6 Supplementary Information .............................................................................91 Chapter 6 – Summary and Future Directions ............................................................... 106 References .................................................................................................................. 108 vi List of Figures Figure 1. Ca transport in ventricular myocytes. ............................................................... 2 Figure 2. Structure of SERCA in E2 state. ...................................................................... 5 Figure 3. SERCA enzymatic cycle. ................................................................................
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