Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Benjamin R. Nixon Graduate Program in Biomedical Sciences The Ohio State University 2014 Dissertation Committee: Brandon J. Biesiadecki, Ph. D. (Advisor) Jonathan P. Davis, Ph. D. Michael A. Freitas, Ph. D. Mark T. Ziolo, Ph. D. Copyright by Benjamin R. Nixon 2014 Abstract Heart disease, with an anticipated $316 billion in economic expenses, affects one in three adults and is the leading cause of death in the United States. In the diseased heart a multitude of cellular changes occur, either as a compensatory mechanism to deter the modifications brought on as a result of cardiac disease or a direct result of the pathophysiology. One such avenue for uncovering the molecular mechanisms underlying cardiac disease and their functional changes is to study post-translational modification (PTM) of proteins. While extensive work has been done characterizing phosphorylation of cardiac contractile regulatory proteins, this work has been conducted investigating the modifications in isolation. Despite the fact that a single phosphorylation site may be sufficient to alter function, the additive functional effect of multiple phosphorylation sites to crosstalk inter-molecularly and change function differently than that evoked by a single phosphorylation must be taken into consideration. Initially, we sought to determine if the metabolic regulatory kinase AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrate that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with ii controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo- phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA- dependent increases in length dependent activation. We next wanted to investigate the effect of ischemic pH on Ser-150 and Ser- 23/24 phosphorylation. We demonstrate phosphorylation of cTnI is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia is known to decrease intracellular pH directly resulting in depressed Ca2+ binding to Tn and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations in ischemia we measured the individual effects of TnI Ser-150 (S150D), Ser-23/24 (S23/24D) or their combined (S23/24/150D) pseudo- phosphorylation on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca2+ binding of all TnIs, TnI S150D attenuated this decrease such that it was similar to non- phosphorylated TnI at normal pH. The dissociation of Ca2+ from troponin C (TnC) was unaltered by pH, such that TnI S150D remained slow, S23/24D remained accelerated and the combination of Ser-150 and Ser-23/24 pseudo-phosphorylation on the same TnI molecule retained accelerated dissociation. Lastly, to investigate the remaining component of thin filament regulation, tropomyosin nitration or phosphorylation was investigated to determine the effect of PTM on structure and function. To investigate the kinetic regulatory role of αTm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild- iii type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm's regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to TnC in reconstituted thin filaments did not differ between the three Tm's, however dissociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. Additionally, we sought to investigate the effect of reactive nitrogen species to nitrate Tm Tyr residues, its structure-function impact and develop a mass spectrometry approach to identify Tm 3-nitotyrosin (3-NT) PTM. Our data demonstrates the pathologically relevant reactive nitrogen species peroxynitrite modifies Tm Tyr residues to 3-NT with structural impact significant to modulate Tm function. We further developed and validated a novel and highly versatile target-driven MS/MS strategy to facilitate identification and quantification of Tm 3-NT without a priori knowledge of target residue modification. In conclusion, the structural and functional modification of thin filament regulatory proteins described above provide an intricate glimpse into the mechanisms in which the heart can undergo to alter function both in normal physiology and in disease states. Gaining a better understanding of protein PTMs associated with cardiac disease will play a major role in the continued development of therapeutics to treat cardiovascular disease. iv Dedication IN LOVING MEMORY OF JOSEPH AND DORIS COTTER, THEODORE NIXON, & KATHERINE WELLS v Acknowledgments Much of the work completed during my dissertation could not have been done without insight and help from others and I would be remiss not to mention and thank those who continuously directed and assisted me over the years. First and foremost I would like to humbly thank my graduate advisor Dr. Brandon Biesiadecki. Brandon’s experience and incessant enthusiasm for his research facilitated my own interest in muscle physiology giving me a fantastic research experience. Furthermore, his invaluable guidance has always taught me to evaluate work critically and maintain my own scientific integrity by not cutting corners when carrying out and conveying my research. Likewise, I would sincerely like to thank Dr. Jonathan Davis, Dr. Mark Ziolo, and Dr. Michael Freitas as their lively conversations and insight as my committee proved to be an aspect instrumental in my research. I would like to thank Dr. Gregory Caputo for not only being likely the best teacher during my time at Rowan University but for being an outstanding lab mentor and fostering my passion for scientific research (as well as frequent Tecmo sessions). Thanks to my advanced chemistry teacher in high school Gerald Biggs for undoubtedly showing me that chemistry, and science in general, is a significantly better career path for me than my initially planned accounting. I cannot thank him enough for kick starting the foundation of interest in science I have today. I would also like to sincerely thank all of the labs and members associated with the Molecular and Cellular vi Cardiophysiology group at The Ohio State University. This group has made presenting scientific work a beneficial and spirited event which I can only hope exists in a similar fashion in my future endeavors. To Elizabeth Brundage and Nathan Hassel in the Biesiadecki lab, it has been a great experience working together and I thank them for all their help both technically and intellectually during my studies in the lab. Many thanks to my fellow Biomedical Sciences graduate program student in the Biesiadecki lab Hussam Salhi who has provided me with numerous conversations discussing our work and the work of the field. Such dialogue has helped guide my thoughts and open my mind to new possibilities. You are going to do good things in the future. A special thanks to my friend that I met while at Rowan University Joe Garofalo for consistently being a great friend and providing me with a way to temporarily forget the stresses associated with completing my doctoral work by means of audiovisual competitions. Lastly, I would like to thank my family and loved ones for their undying support during my time at The Ohio State University. I want to give a special thanks to my loving girlfriend Mellissa Hicks for always offering an ear to talk to and being incredibly supporting and understanding when times were bumpy, albeit because of school or health. I also want to thank my family for continuously supporting me as well as recurrently making the trek out to Columbus from New Jersey to visit. I cannot stress how important it was to be able to see a piece of home frequently during my five years away. All of my successes would not be possible without those mentioned above, and for that I am forever grateful. vii Vita 2005................................................................Palmyra High School 2009................................................................B.S. Biochemistry, Rowan University 2009 to present ..............................................Graduate Research Associate, Department of Physiology and Cell Biology, The Ohio State University Publications 1. Nixon, BR, A Thawornkaiwong, J Jin, EA Brundage, SC Little, JP Davis, RJ Solaro, and BJ Biesiadecki. "AMP-activated Protein Kinase Phosphorylates Cardiac Troponin I at Ser-150 to Increase Myofilament Calcium Sensitivity and Blunt PKA-dependent Function." J. Biol. Chem. 287 (2012): 19136-9147. 2. Nixon, BR,