Integrative Approach to Understanding the Multimodal Effects of Exercise Adaptation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alisa D. Blazek, M.A. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2015 Dissertation Committee: Dr. Noah L. Weisleder, Advisor Dr. Timothy E. Hewett Dr. Sudha Agarwal Dr. Timothy D. Eubank Copyright by Alisa D. Blazek 2015 Abstract The study of the molecular basis of exercise adaptations in the field of exercise physiology is relatively new, as evidenced by the surge in articles citing molecular techniques over the last decade. This focus on molecular indicators is not surprising given the efforts to improve upon preventative healthcare and reduce healthcare cost burden in our country. The ability to exploit molecular indicators of exercise effectiveness as well as discover novel therapeutic options are clear advantages to studying molecular exercise physiology that could impact healthcare. In the studies described here, we used an integrative approach, building from a molecular basis to mice to human subjects, to develop a more comprehensive understanding of the molecular mechanisms mediating the effects of exercise. To study the effects of exercise on a physiological systems-wide level, we used microarray technology to characterize global upregulation and downregulation of genes in response to walking exercise in rat cartilage. We found temporal gene expression changes over 15 days of exercise. The networks of genes affected were responsible for directing extracellular matrix; cell metabolism; cytoskeleton; cell signaling, growth, and differentiation; and inflammatory pathways. It was evident from this study that integration of multiple physiological systems occurs in response to an exercise stimulus. We then aimed to isolate and study selected systems using molecular and physiological techniques. The objective of one particular study was to determine the role ii of exercise as an integrator of bone and muscle health. One of the genes that was observed during the microarray analysis to be upregulated by exercise by more than 1.5 fold was follistatin-like3 (FSTL3). FSTL3 belongs to the follistatin family of molecules which also includes follistatin (FST). Previous studies showed that FSTL3 is required for exercise driven bone formation. This protein also binds and inhibits myostatin, an inhibitor of muscle growth and strength, thus indicating a role for FSTL3 in hypertrophy and force generation. Using muscle contractility assays and genetic knockout mouse models, it was determined that walking exercise, while sufficient for bone growth, was not a potent enough stimulus for improvements in muscle hypertrophy and force generation. The follistatin family of proteins have been shown to be involved in cardiac health, thus we aimed to determine the role of follistatin 288 (FST288), a genetic knockout model for follistatin, in the heart with and without exercise and in response to pressure overload induced by transverse aortic constriction (TAC). We found that knockout of the circulating follistatin mediator, FST315, resulted in reduced hypertrophy in response to TAC, indicating that this molecule likely contributes to the generation of pathological hypertrophy in heart failure. Finally, we aimed to determine the translational potential of our FSTL3 studies in humans. Subjects were exposed to low intensity walking, high intensity walking, or a neuromuscular training program. Bone density, muscular strength, and serum levels of mediators in the follistatin/myostatin system were measured. It was concluded that walking was an insufficient stimulus for increasing FSTL3 to affect bone mass in iii humans, and that FSTL3 was not found to be a suitable biomarker for BMD. However, some gains in muscular strength were observed in previously sedentary patients. These studies show the importance of integration of multiple indicators (molecular, clinical, biochemical) in the study of the physiology of exercise, and they advance a growing field of knowledge. Future mechanistic studies of exercise will increase fundamental understanding that could be exploited to improve health by paving a path for biomarker discovery, developing methods for quantitation of exercise effectiveness, and advancing the possibility of personalized exercise prescriptions and novel therapeutics. iv Dedication This document is dedicated to my husband, family and friends for their patience and understanding. v Acknowledgments I would like to express my sincere gratitude to Dr. Noah Weisleder, my advisor, for his unwavering support, mentorship, and technical guidance throughout the completion of this work. He unselfishly gave of his time and energy, and I would likely not be at this point without his help and encouragement. I would like to thank Dr. Tim Hewett, my co-advisor, for believing in me and having a vision for how this work can fit into a broader, translational role. I realize that he has been a powerful advocate for me over the years, and I am truly grateful and humbled by that. I appreciate his positive energy. Lastly, and certainly not least, I would like to thank Dr. Sudha Agarwal, my co- advisor, for taking a chance on me and finding a way for me to study molecular mechanisms of exercise. I appreciate her hands-on approach in slogging through the day- to-day research with me. I want to thank Dr. Tim Eubank, my committee member, for serving as an unofficial advisor to me when I first started in my graduate program. He has been a sounding board and mentor to me through the years. As this work involves multiple projects across multiple labs, it would not have been possible to perform this research without the assistance of various individuals. vi I am grateful for the contributions of Dr. Zhaobin Xu, Dr. Karthikeyan Krishnamurthy, and Kevin McElhanon, members of the Weisleder lab who performed some of the work presented in the heart and recombinant cell treatment sections of this document. The reproducibility of the contractility work would have suffered without the skilled hands of Eric Beck. Dr. Heather Manring provided valuable assistance in breeding and maintaining some of the mouse models used here. In addition, I would like to thank the other members of the Weisleder lab for being a source of assistance in the lab, as well as being a sounding board for technical and graduate school matters, in particular, Jenna Alloush, Brian Paleo, Travis Gurney, Dr. Liubov Gushchina, Anjella Manoharan, Dr. Sayak Bhattacharya, and Prasanthi Appikatla. I want to express my gratitude to the members of the Hewett lab: Dr. Stephanie DiStasi Roewer has been a mentor and incredible role model for me. Dr. Sam Wordeman and Dr. Nienke Willigenburg provided invaluable technical expertise. Lab managers, Josh Hoffman and Ben Roewer, facilitated this work and were always ready to rescue. I am so grateful to be able to have worked with these five incredibly professional people. Isac Kunnath, Kari Stammen, and Amy Minnema did an exceptional job of recruitment and blood draws. Rachel Tatarski and Albert Chen performed some of the clinical tests in this study. Dr. Jackie Buell did an amazing job with the bone density scans and providing a great experience for the human subjects by taking time with them to explain the results of tests. Aileen Cudia looked out for me and made things happen when others thought it couldn't be done. Samantha Primmer did much for me behind the scenes that has not gone unnoticed. Chris "Godfather" Nagelli has been like a lab brother to me. I vii am so appreciative of the way he responsibly managed my project when I was not able to be physically present as if it were his own. Finally, I want to thank the members of the Agarwal lab. Jackie Li did so much to keep the lab running smoothly, especially mouse breeding, upkeep, and exercising. The mouse experiments presented here could not have happened without her. Jin Nam provided much of the data in the microarray section; I am grateful to be able to carry on his careful research. Although their time in the lab with me was limited, Meera Predhan, Michelle Williams, and Priyangi Perera provided wonderful assistance and friendship. Dr. Derrick Knapik helped to me integrate into the lab and taught me much. His assistance was invaluable. I am appreciative to Ziyue Chen for her assistance with raw data analysis, statistics, and interpretation. I want to thank the subjects who volunteered their time to participate in the study. I appreciate their interest, enthusiasm, and desire to make healthy changes in their lives. The human subjects research in these studies was supported by a Doctoral Student Research Grant from the American College of Sports Medicine. The molecular studies were supported by the Sigma Xi Grants in Aid of Research and The Ohio State University Alumni Grants for Graduate Research and Scholarship. Without the support of these funding agencies, the research would not have been possible. viii Vita 1996................................................................B.A. Zoology, Ohio Wesleyan University 2010................................................................M.A. Exercise Science, The Ohio State University, Columbus, OH 1996................................................................Sales Representative, Laboratory Solutions, Columbus, OH 1997-1999 ......................................................Chemist,