Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 2020 Role of ATP1A1 in Skeletal Muscle Growth and Metabolism Laura C. Kutz [email protected] Follow this and additional works at: https://mds.marshall.edu/etd Part of the Medical Biophysics Commons, Medical Physiology Commons, and the Musculoskeletal, Neural, and Ocular Physiology Commons Recommended Citation Kutz, Laura C., "Role of ATP1A1 in Skeletal Muscle Growth and Metabolism" (2020). Theses, Dissertations and Capstones. 1267. https://mds.marshall.edu/etd/1267 This Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected], [email protected]. ROLE OF ATP1A1 IN SKELETAL MUSCLE GROWTH AND METABOLISM A dissertation submitted to the Graduate College of Marshall University In partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biomedical Research by Laura C. Kutz Approved by Dr. Zijian Xie, Committee Chairperson Dr. Sandrine Pierre, Committee Co-Chairperson Dr. Nalini Santanam Dr. Joseph Shapiro Dr. Judith Heiny Marshall University May 2020 APPROVAL OF THESIS ii DEDICATION This dissertation is dedicated to my advisor and mentor, Dr. Zijian Xie, who passed away before it could be published. I am proud to have been mentored by such a brilliant researcher, and I hope to honor his legacy of hard work, compassionate mentoring, and innovative research as I build my own career on the principles I learned from him and applied to this body of work. iii ACKNOWLEDGEMENTS All of the hard work that went into this dissertation would not have been possible without the support of my friends and family, especially my parents Mike and Rose Mary, my brother Joey, my grandparents Mary Jo and Silas Hannah and Sally and Ralson Kutz, and my entire extended family. I also want to thank my partner Will Johnson and the entire Johnson family for their ongoing support. iv TABLE OF CONTENTS Approval of Thesis ..................................................................................................................... ii Dedication ................................................................................................................................. iii Acknowledgements ................................................................................................................... iv List of Tables ........................................................................................................................... vii List of Figures ......................................................................................................................... viii Abstract ......................................................................................................................................x Chapter 1 ....................................................................................................................................1 Introduction .....................................................................................................................1 Molecular Properties of Na/K-ATPase Isoforms in the Skeletal Muscle ...............2 Regulation of Na/K-ATPase in Skeletal Muscle Physiology ............................... 12 A New Role for NKA α1 in Skeletal Muscle Physiology .................................... 18 Chapter 2 .................................................................................................................................. 20 Isoform-specific role of Na/K-ATPase α1 in skeletal muscle ......................................... 20 Abstract ............................................................................................................. 21 Introduction ....................................................................................................... 21 Materials and Methods ....................................................................................... 23 Results ............................................................................................................... 28 Discussion.......................................................................................................... 34 Acknowledgements ............................................................................................ 40 Chapter 3 .................................................................................................................................. 41 Metabolic capacity, endurance and insulin resistance are acquired by mammals via a common mechanism ...................................................................................................... 41 v Summary ........................................................................................................... 42 Introduction ....................................................................................................... 42 Results ............................................................................................................... 45 Discussion.......................................................................................................... 64 Acknowledgements ............................................................................................ 70 Author Contributions ......................................................................................... 70 Materials and Methods ....................................................................................... 71 Chapter 4 .................................................................................................................................. 81 Discussion and Conclusions........................................................................................... 81 Isoform-specific role of NKA α1 in skeletal muscle ........................................... 81 NKA α1 regulates muscle growth ....................................................................... 81 NKA α1 regulates muscle metabolism................................................................ 83 Evolutionary implications .................................................................................. 84 Clinical implications .......................................................................................... 86 Limitations ......................................................................................................... 88 References ................................................................................................................................ 89 Appendix A............................................................................................................................. 113 Letter from Office of Research Integrity ...................................................................... 113 Appendix B ............................................................................................................................. 114 Abbreviations .............................................................................................................. 114 vi LIST OF TABLES Table 1: NKA α isoform enzymatic properties ............................................................................3 Table 2: Selected physiological parameters in Na/K-ATPase α1 +/+ vs α1 +/- mice. ................. 30 Table 3: Conservation of the NKA α1/Src binding sites in mammals. Bold letters indicate residues that differ from mammalian α1. ................................................................................... 46 Table 4. Amino acid sequences of the Src binding sites of the WT rat α1, WT rat α2, and gain-of- function signaling mutant α2 NKA expressed by the AAC-19, LX-α2, and LY-a2 cell lines. .... 47 Table 5: Volumes and concentrations of injections for Seahorse analysis, with final concentrations in parentheses. ................................................................................................... 73 Table 6: Primer sequences used in RT-qPCR............................................................................. 75 vii LIST OF FIGURES Figure 1: NKA subunits and Albers-Post mechanism ..................................................................4 Figure 2: Schematic diagram of α and β subunits of NKA ...........................................................7 Figure 3. Na/K-ATPase α-isoform abundance in skeletal muscles from Na/K-ATPase α1 haplodeficient mice (α1+/-) and control littermates (α1+/+). ...................................................... 29 Figure 4. Na/K-ATPase activity. ............................................................................................... 31 Figure 5. Changes in muscle mass in α1+/- mice. ...................................................................... 32 Figure 6. Na/K-ATPase α-isoform abundance in skeletal muscles from C57Bl6 mice. .............. 33 Figure 7. Structural changes in the soleus muscle of Na/K-ATPase α1 haplodeficient mice. ...... 34 Figure 8. A. GSK3β and glycogen content in the soleus muscle of Na/K-ATPase α1 haplodeficient mice. .................................................................................................................. 37 Figure 9. Distance to fatigue in Na/K-ATPase α1 haplodeficient mice (α1+/-) and control littermates (α1+/+) during forced treadmill running. .................................................................. 38 Figure 10: Evolutionary relationships