Bartman Dissertation 073018 for Graduate
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MECHANISMS OF CIRCADIAN RHYTHM PROTEIN PERIOD2 IN CARDIOPROTECTION by COLLEEN MARIE BARTMAN B.A., College of Wooster, 2012 A dissertation submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cell Biology, Stem Cells, and Development Program 2018 This dissertation for the Doctor of Philosophy degree by Colleen Marie Bartman has been approved for the Cell Biology, Stem Cells, and Development Program by Julie Siegenthaler, Chair Peter Buttrick Lori Walker Bruce Appel Amanda Law Tânia Reis Tobias Eckle, Advisor Date: August 17th, 2018 ii Bartman, Colleen Marie, Ph.D., Cell Biology, Stem Cells, and Development Program Mechanisms of Circadian Rhythm Protein Period2 in Cardioprotection Dissertation directed by Professor Tobias Eckle ABSTRACT Throughout evolutionary time, all organisms and species on Earth evolved with an adaptation to consistent oscillations of sunlight and darkness, now recognized as ‘circadian rhythm.’ Single-cellular to multi-system organisms use circadian biology to synchronize to the external environment and provide predictive adaptation to changes in cellular homeostasis. Dysregulation of circadian biology has been implicated in numerous prevalent human diseases and therefore targeting the circadian machinery may provide innovative preventative or therapeutic treatment strategies. Previous studies from our lab included a wide search for ischemic preconditioning mechanisms of cardioprotection and identified the light-elicited circadian rhythm protein Period2 (PER2) to be cardioprotective from conditions of low oxygen availability, like myocardial ischemia. However, underlying mechanisms of PER2 dependent cardioprotection remained widely unknown. Our goal moving forward was to decipher the molecular nature of PER2 mediated cardioprotection and use these mechanisms to target PER2 to reduce cellular or tissue damage from hypoxia or ischemia, respectively. We uncovered a multi-faceted role for PER2, functioning both transcriptionally in the nucleus and post-translationally in the mitochondria. Our studies point toward PER2 as a master regulator of metabolic adaptation to hypoxia by working in concert with hypoxia inducible factor 1⍺ and sirtuin 3. Further investigation into ischemic preconditioning mechanisms uncovered the circadian and PER2 dependent microRNA miR-21 to be cardioprotective from ischemia and reperfusion injury. Focused studies into the effects of circadian disruption on the heart revealed that a frequently administered benzodiazepine, midazolam, has deleterious effects on cardiac tissue after ischemia and reperfusion injury. However, circadian amplitude enhancement of PER2 via a novel small molecule inhibitor nobiletin reversed this damage. Moreover, we optimized our daylight regime to iii achieve optimal cardioprotection in a PER2 dependent manner and our mechanistic studies identified downstream cAMP signaling pathways as an underlying mechanism. Lastly, our translational studies into healthy human volunteers suggested that light-elicited cardioprotective strategies identified in the laboratory setting may be beneficial in the clinic. Taken together, we identified several mechanisms of PER2 mediated cardioprotection that may provide innovative preventative or therapeutic treatment strategies for patients with myocardial ischemia. The form and content of this abstract are approved. I recommend its publication. Approved: Tobias Eckle iv ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Tobias Eckle for his continued support, openness to my thoughts and ideas, and mentorship. I would also like to thank Dr. Peter Buttrick and Dr. Lori Walker for their contributions and collaborations throughout my graduate studies. In addition, I appreciate my thesis committee, Dr. Buttrick, Dr. Walker, Dr. Bruce Appel, Dr. Amanda Law, and Dr. Tânia Reis for their thoughtful advice and in particular, thanks to Dr. Julie Siegenthaler for being an outstanding committee chair. Furthermore, I would like to thank Dr. John Tentler for his support in my CCTSI pre-doctoral fellowship and my translational studies. I owe significant thanks to Dr. Yoshimasa Oyama, the post-doctoral fellow in the Eckle lab who performed the mouse surgeries. Lastly, I would like to thank Stephanie Bonney for her prior work in the Eckle lab making the stable knockdown cell lines. I would like to acknowledge my two pre-doctoral fellowships that I received during my four years as a student. My second year I received a Colorado Clinical and Translational Sciences Institute (CCTSI) fellowship (TL1 TR001081) that opened doors of opportunity to broaden my research into clinical applications and enhanced my understanding of translational research. My third and fourth years were funded through an American Heart Association (AHA) fellowship (16PRE30510006), which supported my mechanistic studies, the majority of which are the core of my doctoral research and presented in my dissertation. I am thankful for the funding and opportunities allotted by these fellowships. Additionally, I had the opportunity to present my research at many conferences in Colorado, New Mexico, Washington D.C., and Illinois. These conferences were funded in part by my fellowships, Dr. Eckle’s NHLBI funding (5R01HL122472), the Gates Center for Regenerative Medicine, and Dr. Vesna Jevtovic-Todorovic with the Department of Anesthesiology. The University of Colorado Anschutz Medical Campus and the Graduate Program in Cell Biology, Stem Cells, and Development fostered personal and professional growth during my journey as a graduate student. I am so grateful I had the opportunity to be a part of such a supportive, inclusive, and engaging community. v TABLE OF CONTENTS CHAPTER I. INTRODUCTION ..................................................................................................................... 1 1.1 The Circadian-Hypoxia Link ........................................................................................... 1 1.1.a Cyanobacteria and the Great Oxygenation Event ....................................................... 1 1.1.b Light-sensing pathways: the mammalian circadian system and feedback loop............ 2 1.1.c Oxygen-sensing pathways: hypoxia inducible factors .................................................. 4 1.1.d PAS domains: an evolutionarily link between circadian and hypoxia pathways ........... 5 1.2 PER2 and HIF1⍺ in Metabolic Adaptation to Myocardial Ischemia .............................. 6 1.2.a Adenosine and ischemic preconditioning link PER2 and HIF1⍺ .................................. 6 1.2.b PER2 regulation of HIF1⍺ ........................................................................................... 8 1.2.c Regulation of energy metabolism during hypoxia or ischemia ..................................... 9 1.2.d HIF1⍺ mediated metabolic adaptation to hypoxia ...................................................... 11 1.2.e PER2 and metabolic regulation ................................................................................. 12 1.2.f Overlap between PER2 and HIF1⍺ in metabolic regulation ........................................ 16 1.3 MicroRNAs in Circadian Biology and Therapies ......................................................... 17 1.3.a MicroRNAs as cardiovascular therapeutic targets ..................................................... 17 1.3.b Circadian microRNAs ................................................................................................ 19 1.4 Circadian Disruption at the Root of Disease ............................................................... 21 1.4.a Circadian disruption is associated with a multitude of prevalent diseases ................. 21 1.4.b ICU settings and anesthetics have severe effects on the circadian system ............... 23 1.5 Circadian Considerations in Therapeutic Strategies .................................................. 25 1.5.a Maintaining a robust circadian amplitude .................................................................. 25 1.5.b Diurnal versus nocturnal considerations .................................................................... 26 vi 1.5.c Pharmacological timing and targeting of the circadian system ................................... 27 CHAPTER II. PER2 MEDIATED ENERGY METABOLISM IN ADAPTATION TO HYPOXIA ..................... 39 2.1 Rationale ........................................................................................................................ 39 2.2 Results ........................................................................................................................... 40 2.2.a PER2 transcriptionally controls HIF1⍺ dependent glycolysis during hypoxia ............. 40 2.2.b PER2 is a post-translational regulator of TCA cycle activity during hypoxia .............. 41 2.2.c PER2 regulates mitochondrial function via HIF1⍺-COX4.2 in hypoxia ....................... 42 2.3 Discussion ..................................................................................................................... 43 CHAPTER III. PER2 MICRORNA TARGETS IN METABOLIC ADAPTATION TO HYPOXIA .................... 79 3.1 Rationale ........................................................................................................................ 79 3.2 Results ........................................................................................................................... 79 3.2.a Differential and PER2 dependent