Sphingolipids Modulate the Inflammatory Response and Muscle Function in Mdx Mice Jonathan Adam Doering Thesis to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Human Nutrition, Foods, and Exercise Robert W. Grange, Committee Chair Eva M. Schmelz Klaus D. Elgert June 20, 2013 Blacksburg, Virginia Keywords: Duchenne Muscular Dystrophy, mdx, inflammation, sphingolipids, muscle function Sphingolipids Modulate the Inflammatory Response and Muscle Function in Mdx Mice Jonathan Adam Doering Abstract Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P<0.0001). At 11 wks, only WT+S EDL stress in vitro was greater than all other groups at 50-150 Hz. The in vitro relative stress-frequency relationship of mdx+S EDL was left shifted from the other treatment groups. Inflammatory genetic markers were increased in mdx+S mice. These data suggest treatment of mdx mice with 0.1% SM improves ankle flexor torque in vivo, causes a left shift of the stress-frequency relationship in vitro, and modulates the inflammatory gene signature. Acknowledgements “There is nothing either good or bad, but thinking makes it so”-William Shakespeare First and foremost, I would like to thank my family: my parents, Mark and Gretta Doering for their unconditional love and support through all the challenges and difficulties throughout my life, especially when times were the worst (and all the money that came with the challenges!); my brother Andrew, for being a best friend, rival and someone I can always talk with; and lastly, my brother Bradley for being a constant inspiration and such an integral part of my pursuits in academia. Next, I would like to thank my committee: my teacher, advisor, and friend Dr. Robert Grange for not only giving me the opportunity to pursue my goals, but for always providing the right push and advice when I needed it the most (and your seemingly unlimited patience!); Dr. Eva Schmelz, for your great insights and instruction on laboratory techniques and your great sense of humor which always cheered me up when things were difficult. Finally, Dr. Klaus Elgert for instilling in me your appreciation for scientific inquiry and exploration, as well as your kind suggestions and advice. My thanks to all of you for your patience and support during my studies! Special thanks go to all the undergraduate students who have assisted in the lab. Alex, Becky, Sarah, Greg, Brittany (you know who you are), you made the days pass quickly and your assistance with all the procedures (especially that microarray analysis) was invaluable and it was truly a pleasure to get to know each of you and your goals. I hope to watch as you too develop your personal careers. Special thanks go to Michele Lewis, my first and only TA advisor during my thesis studies. I learned much during my time as your TA and I greatly appreciated the opportunities you provided me to teach class and interact with students. It was a wonderful experience that I will carry fondly through my academic career. Finally, and most importantly, I would like to thank my wonderful wife, Nichole. You have stood by me and supported me through each step of this process. You never let me quit and you continue to push me every day to be a better person. This first year of marriage has been a wonderful experience, and I cannot wait for our next stage in life! iii Table of Contents Abstract....................................................................................................................................... ii Acknowledgements .................................................................................................................... iii List of Figures ........................................................................................................................... vii List of Tables ............................................................................................................................. ix Chapter 1: Introduction ............................................................................................................... 1 Statement of Problem ............................................................................................................. 3 Specific Aims .......................................................................................................................... 3 Main Hypotheses .................................................................................................................... 4 Specific Null Hypotheses ........................................................................................................ 4 Chapter 2: Literature Review ...................................................................................................... 6 Section A: Fundamental Contractile Properties .......................................................................... 6 A.1- Introduction ..................................................................................................................... 6 A.2- Skeletal Muscle Hierarchy ............................................................................................... 6 A.3- Spatial Arrangement of Filaments ..................................................................................10 A.4- Muscle Contraction ........................................................................................................10 A.4.1- Excitation-Contraction Coupling and Muscle Contraction .........................................11 A.4.2- Fiber Types .............................................................................................................13 A.5- Skeletal Muscle Contractile Properties ...........................................................................15 A.5.1- Length-Tension Relationship ...................................................................................15 A.5.2- Twitch, Tetanus, and Force Frequency ....................................................................17 A.5.3- Force-Velocity .........................................................................................................19 A.5.5- Fatigue and Fatigue Recovery .................................................................................20 A.6- Summary .......................................................................................................................21 Section B: Duchenne Muscular Dystrophy ................................................................................22 B.1-Introduction .....................................................................................................................22 B.2- Clinical Presentation ......................................................................................................22 B.3- Dystrophin ......................................................................................................................23 B.4- Dystrophin-Glycoprotein Complex ..................................................................................24 B.5- Pathophysiology of DMD ................................................................................................26 B.6- Animal models of DMD ..................................................................................................27 B.7- Hypotheses of DMD Pathophysiology ............................................................................28 B.7.1- The Mechanical Hypothesis .....................................................................................28 iv B.7.3- Calcium Hypothesis .................................................................................................29 B.7.4- Additional Hypotheses .............................................................................................29 B.8- Summary .......................................................................................................................30 Section C: Inflammatory Response ...........................................................................................30 C.1- Introduction ....................................................................................................................30 C.2- Basics of Inflammation ...................................................................................................31 C.2.1- Cellular Component .................................................................................................31