Contribution of ICAM-1 to the Immunobiology of Skeletal Muscle Hypertrophy
Total Page：16
File Type：pdf, Size：1020Kb
A Dissertation entitled Contribution of ICAM-1 to the Immunobiology of Skeletal Muscle Hypertrophy by Christopher L. Dearth Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Exercise Science ______________________________________________________ Dr. Francis X. Pizza, Committee Chair ______________________________________________________ Dr. Thomas J. McLoughlin, Committee Member ______________________________________________________ Dr. Douglas W. Leaman, Committee Member ______________________________________________________ Dr. Anthony Quinn, Committee Member ______________________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2011 Copyright 2011, Christopher L. Dearth This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Contribution of ICAM-1 to the Immunobiology of Skeletal Muscle Hypertrophy by Christopher L. Dearth Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Exercise Science The University of Toledo May 2011 Our laboratory recently demonstrated that β2 integrins, adhesion molecules expressed by myeloid cells, contribute to the hypertrophic response to muscle overload. The present study was conducted to gain insight into the possibility that β2 integrins promote hypertrophy via a mechanism that is dependent on the expression of ICAM-1, a major ligand for the β2 integrin CD11b/CD18. We found that muscle overload increased gene and protein expression of ICAM-1 and induced ICAM-1 expression by both myofibers and satellite cells/myoblasts via a β2 integrin independent mechanism. Functionally, in-vitro studies demonstrated that activation of ICAM-1 promotes the events of proliferation and hypertrophy in skeletal muscle cells. These results are supported by additional in-vivo experiments which demonstrated that ICAM-1 contributes to skeletal muscle hypertrophy as indicated by greater elevations in muscle mass, myofiber size, and protein content in wild type compared to ICAM-1-/- mice after muscle overload. The iii cumulative interpretation of the results presented herein give credence to our working model which hypothesized that expression of ICAM-1 by skeletal muscle cells after mechanical loading serves as a mechanism by which neutrophils and macrophages can bind to and directly communicate with skeletal muscle cells to promote hypertrophy. iv For Pops, Eric, Cynthia, and Alicija: I dedicate this work to you. Pops – I do not have words to adequately describe my deep gratitude for all of the love, support, and encouragement that you have provided over the years. Thank you for showing me the true meaning of work ethic and unwavering dedication to family. Thank you for instilling in me the belief that anything is possible with hard work. You have shaped me into the person that I am today and for that I am forever grateful. Eric & Cynthia – I could not ask for a better brother or sister. I am deeply appreciative of your love and steadfast support throughout the years. I owe you both an immeasurable debt for all that you have done to help me get to where I am today. Your contributions and guidance mean more than you know. Thank you for everything. Alicija – You and I have shared many burdens, anxieties, and pleasures during this journey. Your support, encouragement, patience, and unwavering love are undeniably the bedrock upon which this document is built. I look forward to showing my appreciation with a lifetime of love, happiness, and wine. Thank you. I love you. v Acknowledgements Advisor: Dr. Francis X. Pizza, thank you. It cannot be overstated how appreciative I am for all that you have done for me in the last 5 years. Your sage advice, patient encouragement, immense knowledge, and infectious enthusiasm truly embody what a mentor should be. You have contributed to my development as a scientist in innumerable ways. I will always consider myself your student and look forward to learning from you for years to come. Committee members: Drs. Thomas J. McLoughlin, Douglas W. Leaman, and Anthony Quinn: thank you for all that you have done during my time at The University of Toledo. Your valuable insight and expertise greatly contributed to the completion of this dissertation. Colleagues: Dr. Jennifer Peterson, Dr. Joseph Marino, Dr. Mitchell Stacy, and Qingnian Goh: your countless time and effort was greatly needed and deeply appreciated. Mitzi & Bruce: thank you for all that you do to keep the department running smoothly. I am grateful to everyone in the department who has made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever. Funding: This project was partially funded by The American College of Sports Medicine; NASA Space Physiology Research Grant. vi Table of Contents Abstract iii Acknowledgements vi Table of Contents vii List of Figures xi 1. Introduction 1 1.1 Significance 1 1.2 Background 2 1.3 β2 integrins 3 1.4 Intercellular Adhesion Molecule-1 (ICAM-1) 5 1.5 β2 integrin-ICAM-1 Interaction in Skeletal Muscle Hypertrophy 7 1.6 Specific Aims 10 2. Methodology 12 2.1 Specific Aim 1 12 2.1.1 Animals 12 2.1.2 Surgical Procedures 13 2.1.3 Muscle Collection 13 2.1.4 Total Protein Extraction / Quantification 14 vii 2.1.5 Myofiber Cross Sectional Area 14 2.1.6 Total RNA Isolation 15 2.1.7 Reverse Transcription 16 2.1.8 Real-time PCR 16 2.1.9 Western Blot Analysis 16 2.1.10 Confocal Microscopy 17 2.1.11 Satellite Cell / Myoblast Isolation 18 2.1.12 Flow Cytometry 18 2.1.13 Statistical Analyses 19 2.2 Specific Aim 2 19 2.2.1 Overview of Experimental Design 19 2.2.2 Stable Transfection Procedure 20 2.2.3 Activation of ICAM-1 Signaling 21 2.2.4 Proliferation 22 2.2.5 Differentiation 23 2.2.6 Hypertrophy 25 2.2.7 Statistical Analyses 26 2.3 Specific Aim 3 27 2.3.1 Animals 27 2.3.2 Surgical Procedures 27 2.3.3 Muscle Collection 28 2.3.4 Total Protein Extraction / Quantification 28 viii 2.3.5 Histology 29 2.3.6 Myeloid Cell Accumulation 29 2.3.7 Statistical Analyses 30 3. Results 31 3.1 Specific Aim 1 31 3.1.1 ICAM-1 is expressed in hypertrophying skeletal muscle in vivo 31 3.1.2 β2 integrins do not contribute to ICAM-1 expression by skeletal muscle cells 34 3.2 Specific Aim 2 34 3.2.1 Skeletal muscle cells do not constitutively express ICAM-1 34 3.2.2 Stable transfection of pβA-ICAM-1 35 3.2.3 Overexpression of ICAM-1 does not influence proliferation 36 3.2.4 Activation of ICAM-1 enhances proliferation 37 3.2.5 Overexpression of ICAM-1 does not influence differentiation 38 3.2.6 Activation of ICAM-1 does not influence differentiation 38 3.2.7 Overexpression of ICAM-1 enhances markers of skeletal muscle hypertrophy 39 3.2.8 Activation of ICAM-1 does not influence skeletal muscle hypertrophy 40 3.3 Specific Aim 3 40 3.3.1 ICAM-1 contributes to overload-induced skeletal muscle hypertrophy in vivo 40 ix 3.3.2 ICAM-1 alters myeloid cell accumulation in overloaded skeletal muscle 41 4. Discussion 42 4.1 Specific Aim 1 42 4.2 Specific Aim 2 47 4.3 Specific Aim 3 54 4.4 Conclusion 56 References 86 A Appendix: Institutional Animal Care & Use Committee Form 114 x List of Figures Figure 1-1: Working model for how β2 integrin-ICAM-1 interactions could contribute to the hypertrophic response to mechanical loading: Cytokines 58 Figure 1-2: Working model for how β2 integrin-ICAM-1 interactions could contribute to the hypertrophic response to mechanical loading: ICAM-1 signaling 59 Figure 2-1: In vitro image capture pattern 60 Figure 3-1: ICAM-1 gene expression in wild type mice 61 Figure 3-2: ICAM-1 protein expression in wild type mice 62 Figure 3-3: Localization of ICAM-1 in control wild type muscle using multi-photon confocal microscopy 63 Figure 3-4: Localization of ICAM-1 in overloaded wild type muscle using multi-photon confocal microscopy 64 Figure 3-5: ICAM-1 expression by skeletal muscle myofibers is associated with increased cross-sectional area in vivo 65 Figure 3-6: Mechanical loading induces ICAM-1 expression by satellite cells / myoblasts in vivo 66 Figure 3-7: ICAM-1 gene expression in wild type & CD18-/- mice 67 Figure 3-8: ICAM-1 protein expression in wild type & CD18-/- mice 68 Figure 3-9: Localization of ICAM-1 in overloaded CD18-/- muscle using multi-photon confocal microscopy 69 Figure 3-10: Skeletal muscle cells do not constitutively express ICAM-1 in vitro 70 xi Figure 3-11: Stable transfection of pβA-ICAM-1 71 Figure 3-12: Overexpression of ICAM-1 does not influence skeletal muscle cell proliferation in vitro 72 Figure 3-13: Activation of ICAM-1 enhances skeletal muscle cell proliferation in vitro 73 Figure 3-14: Activation of ICAM-1 increases BrdU incorporation in proliferating skeletal muscle cells in vitro 74 Figure 3-15: Overexpression of ICAM-1 does not influence skeletal muscle cell differentiation/fusion 75 Figure 3-16: Activation of ICAM-1 does not influence skeletal muscle cell differentiation/fusion 76 Figure 3-17: Activation of ICAM-1 increases BrdU incorporation in differentiated skeletal muscle cells in vitro 77 Figure 3-18: Overexpression of ICAM-1 in skeletal muscle cells enhances markers of hypertrophy in vitro 78 Figure 3-19: Activation of ICAM-1 does not influence markers of hypertrophy in vitro 79 Figure 3-20: ICAM-1 contributes to overload-induced skeletal muscle hypertrophy in vivo 80 Figure 3-21: ICAM-1 expression alters