Contribution of ICAM-1 to the Immunobiology of Skeletal Muscle Hypertrophy

A Dissertation

entitled

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

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

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

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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.

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.

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.

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

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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

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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

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

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

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 myeloid cell accumulation in overloaded skeletal muscle 81

Figure 4-1: β2 integrins augment the production of TNF-α in overloaded skeletal muscle 82

Figure 4-2: Induction of ICAM-1 by skeletal muscle cells after mechanical loading in vitro 83

Figure 4-3: ICAM-1 activation-mediated skeletal muscle cell proliferation occurs synergistically with serum components 84

Figure 4-4: Summary figure: Working model – Cytokines & ICAM-1 signaling 85

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Chapter 1

Introduction

1.1 Significance

Physical inactivity, aging, and several disease states have been associated with increased protein degradation and/or decreased protein synthesis which manifests itself as a reduction in total protein content and size of skeletal muscle (atrophy). Deleterious consequences of muscle atrophy include: muscle dysfunction, impaired performance in activities of daily living, a loss of functional independence, increased risk of injury, disability, clinical frailty, and an overall increase in morbidity and mortality 1-3.

Therapeutic and pharmacological strategies to treat muscle atrophy are being intensely investigated in the hopes of improving the health status of the aging population and to reduce directly associated healthcare costs, currently estimated at more than 26 billion dollars annually 4.

One therapeutic approach proven to enhance the size and function of skeletal muscle is resistance exercise training. An acute bout of resistance exercise elevates rates of protein synthesis in skeletal muscle and resistance

training increases the protein content, size, and strength of the affected muscles (hypertrophy). Understanding the mechanisms involved in regulating skeletal muscle hypertrophy is significant because it can be used as a means to combat muscle atrophy and improve quality of life for the elderly and individuals affected by numerous diseases.

1.2 Background

Resistance exercise provides a powerful stimulus for the cellular and molecular events of hypertrophy as well as the accumulation of myeloid cells

(neutrophils and macrophages) in skeletal muscle. Hypertrophy appears to be dependent on both the proliferation of satellite cells and cell signaling for protein synthesis 5-10. Interestingly, neutrophils and macrophages have been reported to accumulate in skeletal muscle when signs of satellite cell proliferation and signaling for protein synthesis are also evident 11.

Emerging evidence indicates that that neutrophils and macrophages contribute to muscle hypertrophy after muscle overload 11-15. The mechanism(s) by which myeloid cells influence cellular and molecular events required of hypertrophy however, remains to be revealed.

The overall objective of the lab is to better understand how the interplay between myeloid cells and skeletal muscle cells regulate responses and adaptations that occur in skeletal muscle after resistance exercise (e.g. hypertrophy). To this end, our laboratory has utilized β2 integrins, which control the function of myeloid cells, as a means by which to study the

contribution of myeloid cells to skeletal muscle hypertrophy 13, 16. Using mice deficient in the beta subunit of all β2 integrins (CD18-/-) we demonstrated that β2 integrins contribute to overload-induced skeletal muscle hypertrophy

13. Briefly, the expression of β2 integrins was found to influence the accumulation profile of neutrophils and macrophages, temporally regulate satellite cell proliferation, and enhance both p70S6k signaling and muscle differentiation; all of which could serve as mechanisms for how β2 integrins contribute to overload-induced skeletal muscle hypertrophy. However, the underlying mechanism(s) by which β2 integrin expressing myeloid cells bind to and communicate with skeletal muscle cells to promote the hypertrophic response is unknown.

1.3 β2 integrins

β2 integrins are exclusively expressed by cells of the hematopoietic lineage such as neutrophils and macrophages 17-19. Structurally, β2 integrins exist as polypeptide dimers when the common β subunit (CD18) is non- covalently bound to one of four α subunits (CD11a, CD11b, CD11c, CD11d) thereby yielding four distinct β2 integrins (e.g., CD11b/CD18) 17. β2 integrins are cell surface transmembrane glycoproteins which encompass an N- terminal extracellular domain, transmembrane segment, and a short C- terminal cytoplasmic tail.

Activation of β2 integrins is a key step in the regulation of myeloid cell adhesion because it increases their affinity for ligands prior to adhesion 20, 21.

Activation of β2 integrins can occur in response to a variety of physiological signals (e.g. cytokines, bacterial products, and complement proteins) 19, 22-25.

Upon activation, β2 integrins undergo a conformational change, increased lateral mobility / rearrangement, and/or increased protein expression on the membrane of myeloid cells 25-28.

β2 integrins function primarily as adhesion molecules which are required for cell-cell communication between myeloid cells and other cell types 24, 29, 30. The functional activities of myeloid cells are controlled by the ligation of membrane bound β2 integrins with one of several ligands (e.g. intracellular adhesion molecule-1 (ICAM-1), compliment protein iC3b, fibrinogen and lipopolysaccharide (LPS)) 18, 19, 31. Binding of the β2 integrin,

CD11b/CD18, to a ligand initiates effector functions in myeloid cells such as phagocytosis, reactive oxygen species (ROS) production, and cytokine production 23, 24, 29-40. β2 integrins have also been shown to be required for the extravasation/accumulation of neutrophils, but not monocytes/macrophages, into skeletal muscle after mechanical loading 13, 16.

From a clinical perspective, the importance of β2 integrins can be seen in patients with leukocyte adhesion deficiency type 1 (LAD 1). LAD1 patients have mutations in the common β2-chain (CD18) which results in failed association with the α-chain (e.g. CD11b) and ultimately the absence of cell surface β2-integrins 24. As a result, the affected patients have impaired myeloid cell function, which ultimately results in recurrent bacterial

infections and death at a young age 24, 41, 42. This devastating pathology of

LAD1 clearly shows the importance of β2-integrins in that it appears there are no other redundant mechanisms which can account for their critical functions.

1.4 Intercellular Adhesion Molecule-1 (ICAM-1)

ICAM-1 serves as the primary ligand for two of the four β2 integrins

(CD11a/CD18 & CD11b/CD18); whereas, other ICAMs (ICAM-2,-3,-4) and related molecules (VCAM) are not ligands for β2 integrins 17. ICAM-1 is constitutively expressed at low levels on hematopoietic (e.g., lymphocytes and monocytes/macrophages) and non- hematopoietic (e.g., vascular endothelial cells) cells 24, 43-47. Furthermore, ICAM-1 expression may be induced by other cell types in response to inflammatory stimuli (e.g., cytokines and free radicals) 24, 48-50 and by mechanical stress (e.g. cyclic strain and fluid shear stress) in endothelial cells 51-58.

The murine ICAM-1 gene is located on chromosome 9 and consists of seven exons and six introns 59. ICAM-1 gene expression is regulated by multiple signaling pathways (e.g. NFκB, JAK/STAT, MAP Kinase, or Protein

Kinase C (PKC)) after treatment with cytokines (e.g. TNF-α, IL-1β, IFN-γ) or

ROS 60-62.

Structurally, the ICAM-1 protein can exist in two forms: membrane- bound (mICAM-1) and soluble (sICAM-1). mICAM-1 consists of five extracellular Ig-like domains, one transmembrane domain and a short

cytoplasmic tail. Motifs within the first and third domains are responsible for binding to CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), respectively 48,

63. In addition to the full length mICAM-1 protein, at least five different alternatively spliced ICAM-1 variants have been identified, each arising from the exclusion of one or more extracellular Ig-like domains 59, 64, 65. It has been shown that ICAM-1 rapidly forms homodimers and that this conformation facilitates more stable adhesion of myeloid cells by increasing binding avidity for β2 integrins 64, 66-70 as well as induces intracellular signaling in leukocytes

64, 71. mICAM-1 is differentially glycosylated depending on the cell type, giving rise to a molecular weight range of 76 – 115 kDa 60, 63, 72-74. The unglycosylated ICAM-1 protein has a molecular weight of 60 kDa 73.

Previous investigators have shown that the cytoplasmic tail of mICAM-1 does not possess any intrinsic tyrosine kinase activity 48, 75, 76; yet, engagement by a ligand results in tyrosine phosphorylation 48, 77. Therefore, it is suggested that the known associations of ICAM-1 with cytoskeletal proteins α-actinin 78 or adaptor molecules such as moesin and ezrin 74, 79, 80 may serve to facilitate activation of multiple downstream signal pathways

(e.g., MAPK, Rho kinase, and PI3K/Akt/mTOR) 29, 30, 48, 74, 80-85.

sICAM-1 is produced by proteolytic cleavage of mICAM-1 by matrix metalloproteinases (MMPs) or caspases 65, 86-90. Additionally, in vitro administration of IL-1β, IL-4, IFN-у, and LPS, but not IL-6, can cause cleavage of mICAM-1 to its soluble form 91. Elevated plasma concentrations

of sICAM-1 have been found in obese patients 92 and are thought to be associated with increased risk for future coronary events 92, 93. Furthermore, sICAM-1 plasma concentrations have been shown to increase acutely following injurious exercise 94.

Functionally, ICAM-1 serves as a transmembrane glycoprotein capable of eliciting numerous cellular functions through cell-cell and cell-matrix interactions. Two of the most well known functions of ICAM-1 are its involvement with β2 integrins in the firm adhesion and subsequent transendothelial migration of neutrophils 17, 39, 95-98, as well as its function as a co-stimulatory molecule for T-cells 73, 99. In addition, ICAM-1 can promote phagocytosis and the production of reactive oxygen species (ROS), cytokines, and chemokines from monocytes/macrophages 85, 99, 100. ICAM-1 can also regulate cell survival, proliferation, and migration in a variety of cell types 75,

101-103.

1.5 β2 integrin-ICAM-1 Interaction in Skeletal Muscle Hypertrophy

A potential mechanism for how β2 integrins might contribute to skeletal muscle hypertrophy involves myeloid cell adhesion to skeletal muscle cells. The suitability of this working model however, requires the discovery of a ligand that is expressed by skeletal muscle cells after mechanical loading that is capable of interacting with β2 integrins.

Because ICAM-1 is a major ligand for β2 integrins, the overarching hypothesis under investigation is whether induced expression of ICAM-1 by

skeletal muscle cells after mechanical loading serves as a mechanism by which β2 integrin-mediated binding of myeloid cells influence cellular and molecular events that promote skeletal muscle hypertrophy. Hypertrophy- inducing cellular signals generated from β2 integrin-ICAM-1 interactions could originate from either myeloid cells (Figure 1-1) or skeletal muscle cells

(Figure 1-2).

Preliminary experiments in our laboratory have begun to test the contribution of β2 integrins to the cytokine profile in hypertrophying muscle and how this contribution influences proliferation and differentiation of skeletal muscle cells (Figure 1-1). This mechanism is currently being investigated in our lab.

An alternative mechanism by which β2 integrin-ICAM-1 interactions could contribute to the hypertrophic response is via activation of ICAM-1 signaling in skeletal muscle cells (Figure 1-2). Ligation of ICAM-1 has been shown to promote cell survival and motility, cytokine production, nitric oxide production, and activate multiple signaling pathways (e.g., MAPK, Rho kinase, and PI3K/Akt/mTOR) in vitro 48, 81-85, 103. ICAM-1 signaling in skeletal muscle cells however, has not been investigated. Interestingly, signaling through these pathways has been shown to be involved in the regulation of proliferation, differentiation, and hypertrophy of skeletal muscle cells 5, 9, 104, 105. Furthermore, we recently reported that the expression of β2 integrins was associated with augmented satellite cell/myoblast

proliferation as well as enhanced p70S6k signaling and muscle differentiation in hypertrophying skeletal muscle 13. Thus, we hypothesize that activation of

ICAM-1 signaling in skeletal muscle cells augments their ability to proliferate, differentiate, and/or hypertrophy. The work described herein will test central elements of our working hypothesis.

Specific Aims

Specific Aim 1: Test the hypothesis that ICAM-1 is expressed by skeletal muscle cells in hypertrophying muscle of mice.

To test this hypothesis, plantaris muscles from wild type (C57BL/6) and β2 integrin deficient (CD18-/-) mice were subjected to 3, 7, or 14 days of mechanical overload via synergist ablation or a control condition. ICAM-1 gene and protein expression in plantaris muscles was measured by real-time

PCR and western blot analysis, respectively. Cellular localization of ICAM-1 in hypertrophying skeletal muscle was revealed via multi-photon confocal microscopy and flow cytometry.

Specific Aim 2: Test the hypothesis that activation of ICAM-1 enhances proliferation, differentiation, and/or hypertrophy of skeletal muscle cells in culture.

To test this hypothesis, we cross-linked ICAM-1 on the surface of murine skeletal muscle cells in order to determine the effect of ICAM-1 mediated signaling on skeletal muscle proliferation, differentiation, and hypertrophy. Myoblast proliferation was analyzed by counting the total number of cells and by BrdU pulse labeling. Muscle differentiation was assessed by morphology (myotube formation) and western blot analysis of myogenin expression. Hypertrophy of skeletal muscle cells was accomplished by quantifying myotube size and the number of nuclei per myotube.

Specific Aim 3: Test the hypothesis that ICAM-1 contributes to overload-induced skeletal muscle hypertrophy in-vivo.

To test this hypothesis, plantaris muscles from wild type and ICAM-1 knock-out mice (ICAM-1-/-) were subjected to 3, 7, or 14 days of mechanical overload via bilateral synergist ablation or a control condition. Skeletal muscle hypertrophy was assessed by quantifying wet muscle mass, myofiber cross sectional area (CSA), and total protein content. Myeloid cell (e.g. neutrophils and macrophages) accumulation was quantified by immunohistochemistry of muscle cross-sections.

Chapter 2

Methodology

2.1 Specific Aim 1

Animals

Two strains of mice, wild type (C57BL/6) and CD18 deficient

(Itgb2tm1Bay; C57BL/6 background), were obtained (Jackson Laboratory, Bar

Harbor, ME, USA). The CD18 deficient (CD18-/-) mice have a hypomorphic rather than a null allele for CD18 which results in a significantly reduced level (2% and 16%) of CD18 expression on resting and activated leukocytes, respectively 98. Furthermore, the CD18-/- mice display normal muscle histology, function, protein content, and myofiber size at baseline 16 and unlike the null mutants, do not display any known pathology nor die prematurely 98. Mice were fed standard laboratory chow and water ad libitum. All experimental procedures were performed on mice at 3-4 months of age (~ 26 g body mass) and have been approved by the Institutional

Animal Care and Use Committee.

Surgical Procedures

The mice were anesthetized with intraperitoneal injections of 2.5% avertin (tribromoethanol; 0.015 ml/g body mass) with supplemental doses (0.1 ml) given as needed. Muscle hypertrophy was induced using the chronic muscle overload model. Chronic overload of the plantaris muscle was achieved by surgical, bilateral removal of the gastrocnemius and soleus muscles using aseptic techniques. The soleus and gastrocnemius muscles were exposed by making an incision on the posterior-lateral aspect of the lower limb. The distal and proximal tendons of the soleus, lateral and medial gastrocnemius were cut and carefully removed. The plantaris muscle was left intact to function as the primary plantar flexor during normal cage ambulation. Plantaris muscles collected from non-manipulated mice served as a control condition. Sham procedures were not performed because previous work from our lab 13 and others 11 have shown that the surgical procedures associated with the synergist ablation model do not affect any of the outcome measures tested. Incisions were closed with sterile suture and mice recovered from anesthesia on a heated (37°C) platform before being returned to their cage. Mice were free to ambulate for 3, 7, or 14 days.

Muscle Collection

Mice were anesthetized with intraperitoneal injections of 2.5% avertin

(tribromoethanol; 0.015 ml/g body mass) with supplemental doses (0.1 ml)

given as needed. Anesthetized mice were sacrificed after tissue collection via cervical dislocation.

Plantaris muscles were dissected out, blotted dry, and weighed.

Muscles to be used for cross-sectional area analyses were excised, weighed, coated with optimal cutting temperature compound (Fisher Scientific), frozen in melting isopentane cooled on dry ice, and stored at -80°C. Muscles intended for protein expression analyses were frozen in liquid nitrogen, whereas muscles intended for gene expression analysis were treated with

RNALater (Qiagen) and subsequently stored in liquid nitrogen.

Total Protein Extraction / Quantification

Muscles were homogenized in reducing sample buffer (2% sodium dodecyl sulphate, 1.5% dithiothreitol, 1 M Tris-HCl, and 10% glycerol) containing protease inhibitors [1 mM EDTA, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 11 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, and 5 μg/ml sodium orthovanadate]. Homogenates were then centrifuged (5,000 g, 4°C, 10 min), and the amount of protein in supernatants was determined by the filter paper dye-binding assay 106.

Myofiber Cross Sectional Area

Expression of ICAM-1 in hypertrophying skeletal muscle was determined via immunohistochemistry. Transverse sections (10 μm) of muscles were fixed with acetone and then quenched with hydrogen peroxide for 5 min. Following a wash with PBS, the sections were then blocked (3%

bovine serum albumin [BSA], 0.05% Tween 20, and 0.2% gelatin in PBS) for

30 min. Slides were then incubated overnight at 4°C with anti-ICAM-1 antibody (1:50 in PBS; catalog no. AF796; R&D Systems, Minneapolis, MN).

Slides were then washed twice with PBS and then incubated with biotinylated anti-goat IgG (1:200 in PBS; catalog no. 805-065-180; Jackson

ImmunoResearch Laboratories, West Grove, PA) for 30 min. Finally, the sections were incubated with avidin D horseradish peroxidase (1:1,000 in

PBS) for 30 min and subsequently developed with AEC (3-amino-9- ethylcarbazole; Vector Laboratories). Sections were viewed with a light microscope (Olympus IX-70; Olympus, Melville, NY). The cross-sectional area (CSA) of ICAM-1pos and ICAM-1neg myofibers was determined using digital image analysis software (Image Pro Plus).

Total RNA Isolation

Plantaris muscles were pretreated with RNALater (Ambion, Austin,

TX) and homogenized using a bead homogenizer (Tissue Lyser; Qiagen,

Valencia, CA). RNA was then isolated from muscle homogenates using

RNeasy Fibrous Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s specifications. For determination of RNA concentration

(μg/ml), samples were diluted in 10 mM Tris-HCl ph 7.0 in an RNase-free quartz cuvette. A spectrophotometer (Spectronics Instruments Inc.,

Rochester, NY) was used to read the sample absorbance at 260 nm (A260).

RNA purity was determined for each sample by calculating the A260/A280 ratio, with 1.9 – 2.1 being acceptable.

Reverse Transcription

RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., Foster City, CA).

Samples were subjected to one cycle of 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and held at 4°C using the Techne TC-412 thermocycler (Techne, Burlington, NJ). cDNA samples were then stored at -

20°C.

Real-time PCR

Real-time PCR was performed for ICAM-1 using TaqMan PCR master mix and gene expression primers and probes which is an inventory item of

Applied Biosystems: ICAM-1 (assay ID: Mm00516023_m1). Each sample was run in triplicate, and detection was achieved using Applied Biosystems 7500 real-time PCR system. The relative quantification method (ΔΔCT, where CT is threshold cycle) was used to document results, and data was normalized to

GAPDH (assay ID: Mn99999915_g1).

Western Blot Analysis

Homogenized samples were separated on 10% gels via SDS-PAGE.

Proteins were transferred to PVDF-FL membranes (Millipore) using the

Trans-Blot Semidry Transfer cell (Bio-Rad). Ponceau S staining of membranes was done to assess quality of transfer before continuing.

Membranes were blocked with Tris-buffered saline (TBS)/Odyssey blocking buffer (1:1), washed in TBS, and incubated overnight at 4°C with ICAM-1 antibody (R&D Systems; catalog no. AF796) that was diluted 1:500 in TBS-

T/Odyssey blocking buffer (1:1). Membranes were washed in TBS-Tween and incubated with an Alexa Fluor 680 secondary antibody (1:5,000; Invitrogen).

Presence of protein was determined using an Odyssey infrared detection system. Data was expressed in scanning units.

Confocal Microscopy

Transverse sections (25 µm) from the mid-belly of muscles were fixed in 50% acetone/50% methanol, washed with PBS, blocked, and incubated with one or more of the following anti-mouse antibodies or reagent that were diluted in PBS: ICAM-1 (1:20; R&D Systems product # AF796) and/or CD31

(1:50; BD Pharmingen Catalog # 550274). Sections were then washed with

PBS followed by incubation with appropriate fluorescent-conjugated secondary antibody (ICAM-1; Alexa Fluor 488 chicken anti-goat IgG (1:50):

CD31; Alexa Fluor 594 donkey anti-rat IgG (1:50)). Fluorescent wheat germ agglutinin (WGA; Alexa Fluor® 633; 1:500), which recognizes membrane glycoproteins, was used to reveal the membrane of myofibers and other cells residing in plantaris muscles. Sections were mounted with Fluoromount-G

(SouthernBiotech # 0100-20) containing 4',6-Diamidino-2-phenylindole

(DAPI) and analyzed using TCS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems) and the associated software.

Satellite Cell / Myoblast Isolation

Our laboratory isolates satellite cells/myoblasts from plantaris muscles using methods modified from Blau et al 107. Briefly, plantaris muscles of control and overloaded mice were surgically excised, blotted dry and placed in

3 ml of sterile phosphate buffered saline (PBS). The muscles were then mechanically dissociated by mincing the muscle with a razor blade in 3 ml of

Dulbecco's Modified Eagle Medium (DMEM) followed by enzymatic digestion in a solution of 0.1% Pronase (EMD Bioscience) for 1 hour at 37°C with constant mixing. Samples were then centrifuged at 1000g and the pellets were resuspended in growth medium. Pellets were then repeatedly triturated using progressively smaller pipettes to further dissociate the muscles. The supernatant was then passed through a 100µm vacuum filter

(Millipore), centrifuged at 800g to sediment the dissociated cells, resuspended in staining buffer, and counted using a hemocytometer.

Flow Cytometry

ICAM-1 expression by satellite cells in-vivo was assessed by flow cytometry of cells isolated from plantaris muscles. Briefly, plantaris muscles from a control or overloaded mouse were digested and subsequently pooled

(two plantaris muscles from a single mouse is equal to a sample size of one).

Satellite cells/myoblasts were identified by their expression of α7 integrin 108-

110, which is not expressed by fibroblasts 108. To increase the relative detection of satellite cells/myoblasts (integrin α7+ cells), flow cytometry gates were set

on viable cells using propidium iodide, non-leukocytes (CD45neg) and non- endothelial cells (CD31neg). Thus, satellite cells/myoblasts were operational defined as viable cells that were CD45negCD31negintegrin α7+ 109. The total number of satellite cells/myoblasts for each sample was calculated by multiplying the total number of cells isolated from a mouse by the fraction of the total number of cells that were CD45negCD31neg integrin α7+. The total number of cells was then normalized to muscle mass (mg) to control for overload-induced changes in muscle mass.

Statistical Analyses

Using Sigma Stat statistical software (Sigma Chemical, St. Louis,

MO), data sets were analyzed using a one or two way analysis of variance

(ANOVA). The Newman-Keuls post-hoc test was then used to locate the differences between groups when the observed F ratio was statistically significant (p<0.05). Data was reported as means ± SE unless otherwise stated.

2.2 Specific Aim 2

Overview of Experimental Design

Murine skeletal muscle cell lines were used to test the hypothesis that activation of ICAM-1 contributes to proliferation, differentiation, and/or hypertrophy of skeletal muscle cells. The contribution of ICAM-1 expression on proliferation, differentiation, and hypertrophy of skeletal muscle cells was quantified under control conditions and conditions in which ICAM-1

signaling was activated. Activation of ICAM-1 signaling was achieved by antibody cross-linking, an established technique for initiating ICAM-1 signaling 77, 80-83, 111-113. These experiments were conducted using serum containing or serum free cell culture medium.

Because activation of ICAM-1 has been reported to increase the production of soluble factors in various cell types 82, 99, 100, 114-116, we also tested whether activation of ICAM-1 influences the proliferation and hypertrophic response of cultured skeletal muscle cells via an autocrine/paracrine mechanism. This was accomplished by treating skeletal muscle cells with conditioned medium derived from control cultures or cultures in which

ICAM-1 signaling was activated.

Stable Transfection Procedure

C2C12 myoblasts were stably transfected with an expression vector containing murine ICAM-1 under transcriptional regulation of the human β- actin promoter (pHβA-ICAM1; kindly provided by Dr. Stephen Hedrick at

The University of California, San Diego). The pHβAICAM1 plasmid was created by cloning murine ICAM-1 cDNA into the BamH1 restriction site of the pHβAPr-1 expression vector 117. Another population of C2C12 myoblasts were stably transfected with the empty pHβAPr-1 expression vector 118, which was generously provided by Dr. Peter Gunning (The University of New

South Wales). Transfection quality plasmid DNA was prepared in the laboratory of Dr. Douglas Leaman (The University of Toledo, Dept. of

Biological Sciences) using Qiagen midiprep kits and transfected using

Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen).

Briefly, DNA and Lipofectamine™ 2000 were diluted in separate tubes containing Opti-MEM® I Reduced Serum Medium. Next, the diluted DNA was combined with the diluted Lipofectamine™ 2000 in a 1:2 ratio. The complex was then added to the culture dish containing cells and medium.

After 48 hours at 37°C in 5% CO2, the cells were split (1:25) into two dishes containing growth medium and 800 µg/ml G418 (Invitrogen). Cell growth was then observed every 2-3 days at which time fresh G418 containing medium was replaced. After 4 weeks, a portion of the cells were used to ensure proper ICAM-1 protein expression via western blotting, immunofluorescence, and FACS. Once ICAM-1 over-expression was confirmed, the remaining cells were frozen as stocks of ICAM-1+ myoblasts.

Activation of ICAM-1 Signaling

Activation of ICAM-1 signaling was achieved by treating skeletal muscle cells with an anti-ICAM-1 antibody (8µg/ml; YN1/1.7.4; eBioscience; catalog # 16-0541-82) followed by a cross-linking goat anti-rat IgG F(ab')2 IgG antibody (24 µg/ml; Jackson ImmunoResearch Labs; catalog # 112-006-003).

Briefly, the cells were subjected to 3 washes with PBS to remove any residual serum components, followed by 30 min incubation with rat anti-ICAM-1 antibody in DMEM. Any unbound primary antibody was then removed by 3 washes with PBS before a 30 min incubation with goat anti-rat IgG F(ab')2.

All unbound cross-linking antibody was then removed by a final 3 washes with PBS. Cross-linking control experiments consisted of treating the cells with one of the following: primary antibody, primary antibody isotype control, or secondary antibody only.

Proliferation

Muscle cells (C2C12, Empty, or ICAM-1+) were seeded at a density of

2,500 cells/cm2 in 24 well tissue culture plates for 24 hours. During this time, the cells were treated with “growth medium” (DMEM + 10% fetal bovine serum [FBS]), “differentiation medium” (DMEM + 2% horse serum [HS]), or

“serum free medium” (DMEM + 5 µg/mL insulin) 119. After treatment with cross-linking or control antibodies, cells were supplied with an appropriate volume of “basal medium” (DMEM only), “growth medium”, or “serum free medium” for a period of 24 or 48 hours.

Muscle cell proliferation was quantified by counting the number of cells using a hemocytometer and by measuring the incorporation of 5-bromo-

2′-deoxyuridine (BrdU; 100 µM) into the nuclei of cells. Skeletal muscle cells were pulse labeled with BrdU for 2 hours at various time points in order to establish the kinetics of the proliferative response induced by activation of

ICAM-1. For immunofluorescence detection of BrdU, cells were fixed, denatured, and labeled with a rat monoclonal antibody against BrdU (1:500 in PBS, catalog no. G3G4 ; Developmental Studies Hybridoma Bank) followed by a FITC-conjugated donkey anti-rat IgG antibody (1:100 in PBS; catalog no.

115-095-003; Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were then mounted with Vectashield containing DAPI and visualized using an Olympus IX70 fluorescence microscope. BrdU+DAPI+ myoblasts were imaged in 6 standardized fields per well (Figure 2-1). For each field, one picture of BrdU positive cells and one picture of DAPI labeled nuclei was taken and then merged. Digital image analysis software (Image Pro Plus) was used to determine the total number of BrdU positive cells and total number of nuclei in each merged image. Data was expressed as the percentage of BrdU+DAPI+ cells relative to total DAPI+ cells.

Differentiation

Muscle cells (C2C12, Empty, or ICAM-1+) were seeded at a density of

5,000 cells/cm2 and grown to ~90% confluence in “growth medium” in 12 well tissue culture plates. The cells were then subjected to a series of washes to remove any residual FBS. To induce differentiation, cells were incubated for

24 hours in “differentiation medium”. After treatment with cross-linking antibodies, each well was supplied with “differentiation medium” for a period of 48 hours.

Muscle differentiation was assessed by determining the fusion index.

Differentiated muscle cells were viewed at 10X magnification and myotubes defined as myosin heavy chain (MHC) positive cells containing two or more nuclei. To calculate the fusion index, 6 fields were viewed per culture dish in a predetermined manner (Figure 2-1). For each field, one picture of MHC

positive cells and one picture of DAPI labeled nuclei was taken and then merged. Digital image analysis software (Image Pro Plus) was used to determine the total number of nuclei and total number of nuclei within MHC positive cells in each merged image. On average, a total of 8,000 nuclei per field (i.e. 48,000 per well) was analyzed. The fusion index (%) for each field was then calculated as: (Total number of nuclei within myotubes / total number of nuclei) X 100. Fusion indices for the 6 fields in a given well were then averaged and used to represent a sample size of one.

Localization of MHC in cultured skeletal muscle cells was achieved via immunofluorescence. Briefly, wells were washed with PBS, fixed with cold acetone, and then permeabilized with 0.05% triton-x 100 for 10min at room temperature. Cells were then washed with PBS and incubated with blocking buffer for 30min at room temperature. Following PBS washes, cells were incubated with anti-sarcomeric MHC (MF20; Developmental Studies

Hybridoma Bank) diluted 1:20 in blocking buffer for two hours at room temperature. Cells were then washed in PBS, and incubated with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:100;

Jackson Immuno-Research Laboratories). The cultures were mounted with

Fluoromount-G (SouthernBiotech # 0100-20) containing DAPI and analyzed.

Muscle differentiation was also assessed by quantifying the relative abundance of myogenin using western blot analysis, whereas α-Tubulin was used as a loading control. Homogenized samples were separated by 10%

SDS-PAGE using 15 µg of protein per lane. Proteins were then transferred to

PVDF-FL membranes (Millipore) at 20 volts for 1 hour using Towbin’s transfer buffer (10% methanol) and the Trans-Blot Semidry Transfer cell

(Bio-Rad). Membranes were blocked in Odyssey Blocking Buffer (Li-cor) for

30 minutes at room temperature with gentle rocking. Membranes were washed with TBS-T and incubated overnight at 4ºC with anti-Myogenin

(1:250 in 1:1 cocktail of Odyssey Blocking Buffer and TBS-T; clone FD5; BD

Pharminogen) and anti-α-Tubulin (1:1000 in 1:1 cocktail of Odyssey Blocking

Buffer and TBS-T; catalog no. 3873; Cell Signaling Technology) antibodies.

Following several TBS-T washes, membranes were incubated with gentle rocking at room temperature with Alexa Fluor 680 anti-mouse fluorescent secondary antibody (1:10,000 in 1% BSA/TBS-T; catalog no. A21057;

Invitrogen). Membranes were washed with TBS-T followed by TBS and scanned for protein using the Odyssey infrared detection system. Data was expressed in scanning units.

Hypertrophy

Muscle cells (C2C12, Empty, or ICAM-1+) were seeded at a density of 5,000 cells/cm2 and grown to ~90% confluence in “growth medium” in 12 well tissue culture plates. The cells were then subjected to a series of washes to remove any residual FBS. The cells were then induced to differentiate into multinucleated myotubes by the addition of “differentiation medium” for a period of 4 days. At day 4 of differentiation, the cells were then washed to

remove any residual FBS, followed by the antibody cross-linking protocol or a control condition. Each well was then supplied with “differentiation medium” for a period of 48 hours, at which point the cells were then collected and analyzed. The time points of 4-6 days of differentiation were selected for our hypertrophy measures because it is at this time that the process of differentiation (i.e. myotube formation) has subsided and where hypertrophy of myotubes can occur 120.

Indices of myotube hypertrophy (nuclei/myotube, maximum width, and area) were determined using digital image analysis software (Image Pro

Plus). Myotube hypertrophy measurements were made on the same locations of pictures taken for determining the fusion index.

Statistical Analyses

Using Sigma Stat statistical software (Sigma Chemical, St. Louis,

MO), data sets were analyzed using an analysis of variance (ANOVA) using two independent grouping factors (cell type and treatment). The Newman-

Keuls post-hoc test was then used to locate the differences between groups when the observed F ratio was statistically significant (p<0.05). This statistical analysis allowed us to analyze differences between our myoblast cell types. Data was reported as means ± SE unless otherwise stated.

2.3 Specific Aim 3

Animals

Wild type (C57BL/6) mice were obtained from Jackson Laboratory (Bar

Harbor, ME) whereas ICAM-1 knock-out (ICAM1tm1Cws; ICAM-1-/-) were obtained from Dr. C. Wayne Smith (Baylor College of Medicine). ICAM-/- mice were generated by replacing the entire coding region of the ICAM-1 gene with a puromycin cassette 121, which yields no alternatively spliced

ICAM-1 isoforms, as is present with other ICAM-1 mutant mice 59. Mice were fed standard laboratory chow and water ad libitum. All experimental procedures were performed on mice at 12-16 weeks of age (~ 26 g body mass) and have been approved by The University of Toledo Animal Care and Use

Committee.

Surgical Procedures

The mice were anesthetized with intraperitoneal injections of 2.5% avertin (tribromoethanol; 0.015 ml/g body mass). Muscle hypertrophy was induced using the chronic muscle overload model. Chronic overload of the plantaris muscle was achieved by surgical, bilateral removal of the gastrocnemius and soleus muscles using aseptic techniques. The soleus and gastrocnemius muscles were exposed by making an incision on the posterior- lateral aspect of the lower limb. The distal and proximal tendons of the soleus, lateral and medial gastrocnemius were cut and carefully removed.

The plantaris muscle was left intact to function as the primary plantar flexor

during normal cage ambulation. Plantaris muscles collected from non- manipulated mice served as a control condition. Sham procedures were not performed because previous work from our lab 13 and others 11 have shown that the surgical procedures associated with the synergist ablation model do not affect any of the outcome measures tested. Incisions were closed with sterile suture and mice recovered from anesthesia on a heated (37°C) platform before being returned to their cage. Mice were free to ambulate for

3, 7, or 14 days.

Muscle Collection

Mice were anesthetized with intraperitoneal injections of 2.5% avertin

(tribromoethanol; 0.015 ml/g body mass). Anesthetized mice were sacrificed after tissue collection via cervical dislocation.

Plantaris muscles were dissected out, blotted dry, and weighed.

Total Protein Extraction / Quantification

Muscles were homogenized in reducing sample buffer (2% sodium dodecyl sulphate, 50 mM TCEP [Bond-Breaker TCEP Solution; catalog no.

77720; Thermo Scientific], 1 M Tris-HCl, and 10% glycerol) containing protease inhibitors [1 mM EDTA, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 11

mM 4-(2-aminoethyl) benzenesulfonyl fluoride, and 5 μg/ml sodium orthovanadate]. Homogenates were then centrifuged (5,000 g, 4°C, 10 min), and the amount of protein in supernatants was determined by the filter paper dye-binding assay 106.

Histology

Transverse sections (10 µm) were stained with hematoxylin and eosin

(H&E) and examined for signs of injury and regeneration. Myofibers that showed a pale or discontinuous cytoplasmic staining, were substantially swollen in appearance, or were invaded by cells were classified as injured 16,

122. The number of central nucleated myofibers, a measure of regeneration 13,

16, 123, 124, was counted and then expressed as a percentage of the total number of myofibers within each section. The cross sectional area (CSA) of central nucleated and normal myofibers was determined using digital image analysis software (Image Pro Plus). Normal myofibers were defined as those that did not show signs of injury or central nucleation.

Myeloid Cell Accumulation

Transverse sections (10 μm) were fixed in cold acetone and quenched in hydrogen peroxide. Neutrophils were identified using an anti-mouse Ly6G antibody [clone RB6-8C5; 1:100 in phosphate-buffered saline ([PBS]; BD

Pharmingen, Franklin Lake, NJ), whereas macrophages were recognized using an anti-mouse F4/80 antibody (clone CI:A3-1; 1:100 in PBS; Serotec

Inc., Raleigh, NC). Slides serving as negative controls received PBS instead

of primary antibody. Following a 2 hr incubation at room temperature with primary antibody, sections were washed in PBS, incubated with a biotinylated secondary antibody (Vector Laboratories Inc., Burlingame, CA) followed by avidin D horseradish peroxidase. Sections were then developed with 3-amino-9-ethylcarbazole (Vector). Sections were then viewed with a light microscope (Olympus IX-70) with Nomarski optics. Labeled cells in entire sections were manually counted, and the total number of cells expressed relative to the volume of the section area (Ly6G+/mm3 or

F4/80+/mm3).

Statistical Analyses

Using Sigma Stat statistical software (Sigma Chemical, St. Louis,

MO), data sets were analyzed using an analysis of variance (ANOVA) using two independent grouping factors (strain of mice and overload duration). The

Newman-Keuls post-hoc test was then used to locate the differences between groups when the observed F ratio was statistically significant (p<0.05). This statistical analysis allowed us to analyze differences between our overloaded wild type and ICAM-1-/- mice. Data was reported as means ± SE unless otherwise stated.

Chapter 3

Results

3.1 Specific Aim 1

ICAM-1 is expressed in hypertrophying skeletal muscle in-vivo.

We evaluated the temporal expression pattern of ICAM-1 and it’s localization to skeletal muscle cells in overloaded muscles of wild type mice via real-time PCR, western blot analysis, and multi-photon confocal microscopy. Real-time PCR revealed that ICAM-1 gene expression in overloaded muscles was elevated by 28, 21, and 11 fold following 3, 7, and 14 days of overload, respectively (Figure 3-1). Similarly, western blotting analysis of overloaded muscle revealed that total ICAM-1 protein expression was significantly elevated by 4-6 fold in overloaded muscle compared to control muscle (Figure 3-2).

In order to gain insight as to the cellular localization of ICAM-1 in overloaded skeletal muscle, immunofluorescent labeling experiments using multi-photon confocal microscopy were performed. Fluorescent-conjugated wheat germ agglutinin (WGA), which binds to membrane glycoproteins, was

used to visualize the sarcolemma of myofibers and the plasma membrane of other cells 125.

Analysis of control plantaris muscles revealed ICAM-1 expression in cells neighboring myofibers (Figure 3-3). Double immuno-fluorescent labeling and co-localization analysis software revealed that as much as 97% of ICAM-1 labeling in control muscle was co-localized to endothelial cells

(CD31+). This finding is consistent with the constitutive expression of ICAM-

1 by endothelial cells in skeletal muscle 43, 126. Importantly, ICAM-1 was not found to be expressed by myofibers in control muscle.

In overloaded muscles, the percentage of endothelial cells (CD31+) expressing ICAM-1 was similar to that observed in control muscles.

However, following 7 days of overload, ICAM-1 was found to be expressed by mononuclear cells as well as on the sarcolemma of approximately 18% of myofibers, as indicated by the co-localization of ICAM-1 and WGA (Figure 3-

4). Labeling experiments of 14-day overloaded muscle also revealed ICAM-1 expression by approximately 20% of mature myofibers (Figure 3-4).

Interestingly, cross-sectional area analysis of 14-day overloaded muscle revealed that ICAM-1+ myofibers were 25% larger than myofibers that did not express ICAM-1 (Figure 3-5).

Localization experiments indicated that numerous mononuclear cells appear to express ICAM-1. We hypothesized that satellite cells/myoblasts and leukocytes would be among the cell types that express ICAM-1 in

hypertrophying skeletal muscle. Therefore, to substantiate overload-induced expression of ICAM-1 by mononuclear cells, we performed flow cytometry on cells isolated from control and overloaded plantaris muscles.

In control muscles, 16% of the gated cells and 4% of the total number of cells isolated were satellite cells/myoblasts (CD45negCD31negintegrin α7+).

Of these cells, 0.3% of the gated and 0.05% of the total number of cells were

ICAM-1+. Thus, very few satellite cells/myoblasts in plantaris muscles of control mice were found to express ICAM-1 (Figure 3-6 B).

In agreement with our confocal images, flow cytometry analysis showed that satellite cell/myoblast number was increased by 4 fold at 7 d of overload (3054 cells/mg) compared to controls (715 cells/mg). These data are in agreement with our prior work which demonstrated a 4 fold increase in satellite cell/myoblast proliferation at 7 d of overload in wild type mice 13.

The most important finding however, was the expression of ICAM-1 by satellite cells/myoblasts in overloaded muscles. Of the satellite cells/myoblasts found in 7 d overloaded muscles, 2.6% of the gated and 0.5% of the total number of cells were ICAM-1+ (CD45negCD31neg integrin

α7+ICAM-1+). This resulted in a 5 fold higher number of ICAM-1+ satellite cells/myoblasts in overloaded muscles compared to control muscles (Figure 3-

6). These findings demonstrate for the first time the induction of ICAM-1 on satellite cells/myoblasts in hypertrophying muscle.

In addition to endothelial and skeletal muscle cells expressing ICAM-1 in overloaded skeletal muscle, we hypothesized that a portion of the ICAM-1+ mononuclear cells we observed in our confocal localization experiments would be leukocytes. Thus, we confirmed this hypothesis via flow cytometry and found that ~80% of leukocytes (CD45+) express ICAM-1 in overloaded skeletal muscle (data not shown).

β2 integrins do not contribute to ICAM-1 expression by skeletal muscle cells

We also tested the hypothesis that β2 integrins contribute to skeletal muscle cell ICAM-1 expression following mechanical loading. To test this hypothesis, we analyzed overloaded plantaris muscles from β2 integrin deficient mice (CD18-/-). Contrary to our hypothesis, we found similar levels of gene (Figure 3-7) and protein (Figure 3-8) expression of ICAM-1 between wild type and CD18-/- muscle. These results were confirmed by additional localization experiments which revealed that ICAM-1 expression by myofibers was similar between wild type and CD18-/- overloaded muscles

(Figure 3-9).

3.2 Specific Aim 2

Skeletal muscle cells do not constituetively express ICAM-1

Previous investigators have established that cultured human myoblasts or myotubes do not constitutively express ICAM-1 91, 127, 128; yet, it has also been reported that ICAM-1 mRNA and protein levels can be

significantly increased by treating cultured myoblasts and/or myotubes with one or more cytokines (IFN-γ, TNF-α, IL-6, IL-1β, IL-4) 91, 127-130. Our current study extends these observations by demonstrating that cultured murine skeletal muscle cells (i.e. C2C12) do not constitutively express ICAM-1 at any point during the myogenic process (Figure 3-10). However, in agreement with previous reports 91, 127-130, these cells are capable of induced expression of

ICAM-1 by treatment with TNF-α (10 ng/mL for 24 hours) (Figure 3-10).

Stable tranfection of pβA-ICAM-1

Because skeletal muscle cells do not constitutively express ICAM-1; a cell line of skeletal muscle cells that constitutively express ICAM-1 (ICAM-1+ myoblasts) was created. Another population of C2C12 myoblasts was stably transfected with the empty pHβAPr-1 expression vector (Empty Myoblasts).

Immunoblots of cell lysates from a murine macrophage cell line (RAW

264.7 cells) showed a prominent band at 120 kDa (Figure 3-11; Lane 2). As expected, no ICAM-1 was detected in cultures containing either proliferating myoblasts (Figure 3-11; Lane 3) or myoblasts transfected with the empty expression vector (Figure 3-11; Lanes 4 and 5). These observations confirm that cultured myoblasts do not constitutively express ICAM-1 and demonstrate that our empty vector does not induce ICAM-1 expression.

To serve as positive controls, we treated non-transfected myoblasts with TNF-α (10 ng/ml for 24 h) and analyzed them for ICAM-1 expression via flow cytometry and western blot. We found that TNF- α induced ICAM-1

expression on 35% of the cultured myoblasts and resulted in a prominent band appearing at 110 kDa (Figure 3-11; Lane 6). Importantly, transfection of the ICAM-1 plasmid resulted in a dramatic increase in protein levels of

ICAM-1 (Figure 3-11; Lanes 7 and 8). Qualitative observations indicated that

ICAM-1 over-expression in ICAM-1+ myoblasts is not toxic nor does it appear to exhibit any deleterious, non-specific effects on the myogenic program as evidenced by their ability to proliferate and differentiate into myotubes. In conclusion, we successfully created a cell line of ICAM-1+ myoblasts that are capable of differentiating into myotubes. Thus, we are able to test the hypothesis that activation of ICAM-1 signaling in skeletal muscle cells augments their ability to proliferate, differentiate, and/or hypertrophy.

Overexpression of ICAM-1 does not influence proliferation

Preliminary studies evaluated whether overexpression of ICAM-1 augments skeletal muscle cell proliferation. Muscle cells (C2C12, Empty, and ICAM-1+) were grown in culture for up to 3 days in growth medium.

Cells were then collected and counted via a hemocytometer at day 2 or 3. As expected, cell numbers increased approximately 2.5 and 10-fold following 48 and 72 hours in growth medium, respectively (Figure 3-12). Importantly, no differences were seen between cell types in their ability to proliferate in growth medium. These results suggest that the overexpression of ICAM-1 in skeletal muscle cells does not influence their ability to proliferate.

Activation of ICAM-1 enhances proliferation

ICAM-1 on the surface of skeletal muscle cells was activated, via antibody cross-linking, in order to determine the effect of ICAM-1 mediated signaling on skeletal muscle cell proliferation. After treatment with cross- linking or control antibodies, cells were supplied growth medium for a period of 24 hours. Myoblast proliferation was analyzed by counting cells with a hemocytometer (Figure 3-13) as well as BrdU pulse labeling (Figure 3-14).

Cross-linking of ICAM-1+ myoblasts resulted in a significant increase

(~38%) in cell number compared to control cultures containing ICAM-1+ myoblasts (Figure 3-13 A). In order to confirm that the observed proliferative response was specific to antibody cross-linking; several antibody control experiments (e.g. primary antibody, primary antibody isotype control, or secondary antibody only) were preformed. Treatment of ICAM-1+ cells with the antibody controls did not influence myoblast proliferation (Figure 3-13 B).

To test whether the increased number of cells was due to increased proliferation, BrdU pulse labeling was performed (Figure 3-14). BrdU incorporation in control cultures of ICAM-1+ cells averaged ~50% throughout the 24 hour period. Specifically, the percentage of BrdU positive cells was

51%, 48%, and 52% at 2, 6, and 24 hours in growth medium, respectively. In accordance with an increase in total cell number, cross-linking of ICAM-1 increased BrdU incorporation to levels that were significantly higher than those found for the control condition (Figure 3-14). Collectively, these results

suggest that antibody cross-linking of ICAM-1 mediates signaling events that result in enhanced myoblast proliferation, perhaps through an acute regulation of cell cycle kinetics.

Overexpression of ICAM-1 does not influence differentiation

To evaluate whether overexpression of ICAM-1 augments skeletal muscle cell differentiation, muscle cells (C2C12, Empty, and ICAM-1+) were subjected to differentiation medium (DMEM + 2% HS) for up to 3 days. The overexpression of ICAM-1 did not alter the differentiation process as seen by similar myogenin protein expression (Figure 3-15 A & B) and fusion index

(Figure 3-15 C) between the cell types. These results suggest that overexpression of ICAM-1 does not influence the early events of skeletal muscle cell differentiation.

Activation of ICAM-1 does not influence differentiation

We cross-linked ICAM-1 after 1 day in differentiation medium to examine whether activation of ICAM-1 augments skeletal muscle cell differentiation / fusion. All measurements were made at day 3 of differentiation (i.e. 48 hours post cross-linking). Our results show that activation of ICAM-1 did not alter the differentiation process as seen by similar myogenin protein expression (Figure 3-16 A & B) and fusion index

(Figure 3-16 C) between control and cross-linked cultures. However, we found a significant increase in proliferation (~23% increase in BrdU

incorporation) of ICAM-1+ mononuclear cells (i.e. non-differentiated) upon antibody cross-linking in differentiating cultures (Figure 3-17). These findings have the potential to augment our overall interpretations because fusion index is calculated based on total cell number. Therefore, the observed increase in proliferation due to cross-linking could potentially mask a subtle difference in fusion/differentiation. Therefore, additional experiments are needed to more definitively characterize the affect ICAM-1 activation has on differentiation.

Overexpression of ICAM-1 enhances markers of skeletal muscle hypertrophy

To evaluate whether overexpression of ICAM-1 augments skeletal muscle cell hypertrophy, muscle cells (C2C12, Empty, and ICAM-1+) were differentiated for up to 6 days. Our findings indicate that overexpression of

ICAM-1 promotes the events of myotube hypertrophy as seen by significant increases in several markers of hypertrophy in ICAM-1+ cells compared to controls (Figure 3-18 A-C). Specifically, the overexpression of ICAM-1 by skeletal muscle cells resulted in a 54%, 22%, and 69% increase in the average number of nuclei per myotube, maximum myotube width, and total myotube area, respectively, compared to control cultures at day 6 of differentiation.

These results are intriguing and could potentially suggest that ICAM-1 mediated signaling in skeletal muscle cells enhances the cellular and molecular events required for hypertrophy.

Activation of ICAM-1 does not influence skeletal muscle hypertrophy

ICAM-1 on the surface of skeletal muscle cells was cross-linked at day

4 of differentiation in order to determine the effect of ICAM-1 activation on skeletal muscle hypertrophy. All measurements were made at day 6 of differentiation (i.e. 48 hours post cross-linking). Our results show that cross- linking of ICAM-1 in skeletal muscle cells does not promote indices of hypertrophy above those found in control cultures (Figure 3-19 A-C). We interpret these findings to indicate that the evaluation of ICAM-1 activation on skeletal muscle hypertrophy is potentially confounded due to increased basal hypertrophy associated with overexpression of ICAM-1 (Figure 3-18).

3.3 Specific Aim 3

ICAM-1 contributes to overload-induced skeletal muscle hypertrophy in vivo.

Wet muscle mass was significantly higher in overloaded (14 d) wild- type relative to their control counterparts and was higher in wild-type relative to ICAM-1-/- mice at 14 days of overload (Figure 3-20 A). Total protein content (Figure 3-20 B) and myofiber CSA (Figure 3-20 C) was significantly higher in wild-type compared with ICAM-1-/- mice at 14 days of overload. In wild-type mice, total protein content at 14 days of overload was approximately two fold higher than controls. In 14-day overloaded muscles of

ICAM-1-/- mice, total protein was approximately 11% higher than control levels. Thus, the ICAM-1 deficiency blunted the hypertrophic response to muscle overload. Because we did not examine overload time points beyond 14

days, it is unknown whether overloaded ICAM-1-/- mice would eventually show more substantial signs of hypertrophy.

ICAM-1 alters myeloid cell accumulation in overloaded skeletal muscle

In control plantaris muscles, neutrophil concentrations were 5-fold higher, whereas macrophages were unchanged in wild type relative to ICAM-

1-/- mice (Figure 3-21). At 7 days of overload in wild type mice, neutrophils were 50-fold higher than control and 45-fold higher than ICAM-1-/- mice.

At 7 days of overload, macrophages were elevated approximately 230 and 190 fold, compared to control levels in wild type and ICAM-1-/- mice, respectively.

Furthermore, neutrophil concentrations were still significantly elevated in wild type 14-day overloaded muscle relative to both ICAM-1-/- (10-fold) and control (8-fold) muscles. Macrophages remained significantly elevated above control levels in both wild type and ICAM-1-/- mice in 14-day overloaded muscle. In agreement with our previous work 13, we did not find neutrophils or macrophages in the cytoplasm of myofibers at any time point of overload, suggesting that myofiber necrosis is not a prevalent feature of the synergistic ablation model. Collectively, these results suggest that ICAM-1 alters neutrophil, but not monocyte/macrophage, accumulation in overloaded skeletal muscle.

Chapter 4

Discussion

4.1 Specific Aim 1

Our laboratory recently demonstrated that β2 integrins, adhesion molecules expressed by leukocytes, contribute to overload-induced skeletal muscle hypertrophy 13. One potential mechanism by which β2 integrin expressing myeloid cells could influence the hypertrophic response of skeletal muscle involves ICAM-1, the major ligand for the β2 integrin, CD11b/CD18.

We reasoned that if skeletal muscle cells express ICAM-1 in hypertrophying muscle, then this could serve as a mechanism by which β2 integrins promote hypertrophy of skeletal muscle via adhesion-induced activation of myeloid cells and/or via adhesion-induced activation of ICAM-1 signaling within skeletal muscle cells.

Prior investigators have examined the expression of adhesion molecules, such as ICAM-1, by skeletal muscle cells. These studies have demonstrated that human skeletal muscle cells do not constitutively express

ICAM-1 in vitro 91, 127, 128 or in vivo 126, 131-135. In agreement with this prior

work, we also did not find ICAM-1 to be expressed by murine myofibers in control muscles in vivo nor in a murine skeletal muscle cell line (i.e. C2C12) during in vitro myogenesis.

Skeletal muscle cells have the ability to express immunologically relevant adhesion molecules when stimulated with pro-inflammatory stimuli

91, 126-137. As such, previous investigators have established that ICAM-1 mRNA and protein levels can be significantly increased by treating cultured myoblasts and/or myotubes with one or more cytokines (e.g. IFN-γ, TNF-α,

IL-6, IL-1β, IL-4) 91, 127-130. In-vivo myofiber expression of ICAM-1 has also been reported in patients with inflammatory myopathies 126, 131-135 and in a rodent models of myositis 138 and ischemia-reperfusion injury 139; however, no evidence exists on ICAM-1 expression by skeletal muscle cells in non- pathological in vivo conditions.

Interestingly, several cytokines that are known to induce the expression of ICAM-1 (e.g. TNF-α, IL-6, and IL-1β) have been reported to be elevated in hypertrophying skeletal muscle 12, 140. Therefore, our initial objective was to test the hypothesis that mechanical loading-induced hypertrophy of skeletal muscle is associated with increased gene and protein expression of ICAM-1 and its localization to skeletal muscle cells. We hypothesized that induced expression of ICAM-1 by skeletal muscle cells after mechanical loading may serve as a means by which neutrophils and

macrophages can bind to and directly communicate with skeletal muscle cells to promote hypertrophy.

In accordance with our hypothesis, our data reveals that ICAM-1 is significantly up-regulated in and expressed by satellite cells/myoblasts and mature skeletal muscle fibers following mechanical loading. ICAM-1 was found to be expressed by satellite cells/myoblasts and myofibers in overloaded muscles at a time when myeloid cell accumulation, satellite cell/myoblast proliferation, and signaling for protein synthesis are apparent 5-15. Thus, the appearance of ICAM-1+ skeletal muscle cells in overloaded muscles was temporally associated with events that are known to precede and to contribute to mechanical loading-induced hypertrophy. It is intriguing to postulate whether induced expression of ICAM-1 on satellite cells/myoblasts contributed to the temporal regulation of satellite cell proliferation observed in our prior work 13. However, the functional role of ICAM-1 expression by satellite cells/myoblasts in hypertrophying skeletal muscle is currently unclear.

The apparent aggregation/clustering of ICAM-1 on the sarcolemma of myofibers is thought to represent dimerization of ICAM-1 69, 70, which may be physiologically relevant as it is known to enhance binding affinity for β2 integrins 66-69 and be required for ICAM-1 mediated signaling 113, 141.

Furthermore, we found CD18+ myeloid cells that were closely associated with

ICAM-1+ myofibers and that ICAM-1+ myofibers were 25% larger than those

that did not express ICAM-1 at 14 d of overload. We interpret these data to indicate that skeletal muscle cell specific ICAM-1 expression and/or activation is fundamentally important for the hypertrophic response to muscle overload.

Studies investigating muscle biopsies from patients with inflammatory myopathies have shown that ICAM-1 is expressed by normal myofibers as well as those that are classified as regenerating, invaded, and/or necrotic 126,

131, 133, 134, 138. In contrast to these previous observations, all of the ICAM-1+ myofibers observed in the present study were deemed to be normal, as they did not display hallmark features of injury/necrosis (e.g. invaded by myeloid cells) or regeneration (central nucleation). Furthermore, ICAM-1+ myofibers at 14 days of overload showed signs of hypertrophy, not necrosis or regeneration. Thus, it appears that the mechanism for induced ICAM-1 expression by myofibers as well as the function(s) it may perform can vary considerably depending on the stimulus.

We also tested the hypothesis that β2 integrins contribute to skeletal muscle cell ICAM-1 expression following mechanical loading. Recent work has implicated β2 integrins in controlling cytokine production from neutrophils and macrophages. Interestingly, β2 integrins have been found to regulate the production of some of the cytokines that are known to increase

ICAM-1 expression in skeletal muscle cells. For example, β2 integrins have been shown to stimulate the production of interleukin-1β (IL-1β), and tumor

necrosis factor-α (TNF-α) from human neutrophils 34, 39 and interleukin-1β

(IL-1β) from macrophages 38. Conversely, β2 integrins have also been shown to down regulate interleukin-1β (IL-1β), and interleukin-6 (IL-6) production from mouse macrophages 35. Furthermore, preliminary data from our laboratory has shown that β2 integrins contribute to TNF- α production in hypertrophying skeletal muscle (Figure 4-1). Thus, because β2 integrins appear to control cytokine production by myeloid cells, we hypothesized that

β2 integrins contribute to the induction of ICAM-1 on skeletal muscle cells in overloaded muscles. Support for this hypothesis may indicate a feed forward mechanism whereby β2 integrin signaling in neutrophils and macrophages prepares skeletal muscle cells for subsequent interaction with neutrophils and macrophages.

In contrast to our hypothesis, we found similar levels of gene and protein expression/localization of ICAM-1 in overloaded muscles of wild type and CD18-/- mice. These findings indicate that overload-induced ICAM-1 expression occurs via a β2 integrin independent mechanism. Furthermore, we have previously shown that CD18-/- mice showed little to no signs of hypertrophy after muscle overload 13, yet overload-induced elevations in gene and protein levels of ICAM-1 and its expression by myofibers were similar between strains. These findings indicate that induced ICAM-1 expression by skeletal muscle cells per se is not sufficient to promote hypertrophy and that

β2 integrin-ICAM-1 interaction may be necessary for the production of

hypertrophic stimuli after muscle overload. Such that, a deficiency in or an absence of either β2 integrins or ICAM-1 impairs the hypertrophic response to muscle overload.

Alternatively, several in vitro studies have shown that mechanical strain (i.e. cyclic stretch) can increase ICAM-1 expression in a variety of cell types (e.g. endothelial, type 2 alveolar, and smooth muscle cells) 51, 52, 54-57.

Additionally, several cytokines that are known to induce ICAM-1 expression

(e.g. TNF-α, IL-6, and IL-1β) have been reported to be elevated in skeletal muscle after mechanical loading 12, 140. Therefore, we hypothesized that the events associated with mechanical loading of skeletal muscle are responsible for ICAM-1 expression. To this end, our laboratory has conducted pilot experiments in which non-transfected skeletal muscle cells (C2C12) were subjected to mechanical strain in-vitro. These preliminary experiments are promising in that they show significant increases in ICAM-1 expression by skeletal muscle cells subjected to a non-injurious stain protocol (10% strain)

(Figure 4-2). Furthermore, when these cultures are exposed to TNF-α during this protocol, a synergistic induction of ICAM-1 emerges. These results, although preliminary, could provide insight as to the potential mechanism by which ICAM-1 is expressed in overload-induced skeletal muscle hypertrophy.

4.2 Specific Aim 2

Due to ICAM-1 being a transmembrane glycoprotein, ligation by β2 integrin expressing myeloid cells could have direct affects on intracellular

processes. Numerous investigators have used cross-linking antibodies to mimic the interaction of ICAM-1 with its ligand (e.g. β2 integrins) 77, 80-83, 111-

113. These studies have shown that ligation of ICAM-1 induces receptor clustering on the cell surface 80, 113, and cell signaling via MAPK, Rho kinase,

ERK, and PI3K/Akt pathways 48, 77, 82-84. Interestingly, signaling through these pathways has been shown to be involved in the regulation of proliferation, differentiation, and hypertrophy of skeletal muscle cells 5, 9, 104,

105, 142. Furthermore, our lab recently reported that the expression of β2 integrins was associated with augmented satellite cell/myoblast proliferation as well as enhanced p70S6k signaling and muscle differentiation in hypertrophying skeletal muscle 13. Whether adhesion-induced activation of

ICAM-1 by β2 integrins on myeloid cells contributed to these findings remains to be determined. Therefore, we conducted in-vitro experiments to test whether expression and/or ligation of ICAM-1 on the surface of skeletal muscle cells influences key cellular and molecular events that promote skeletal muscle hypertrophy.

Our findings show that overexpression of ICAM-1 by myoblasts does not influence their ability to proliferate in-vitro. However, upon antibody cross-linking, a robust increase in total cell number was observed. These novel observations suggest a role for ICAM-1-mediated signaling in the regulation of skeletal muscle cell proliferation. Previous studies have shown

ICAM-1-mediated proliferation in other cell types. For example, activation of

ICAM-1 induced proliferation of B-lymphoid Raji cells 75 and increased endothelial cell survival through activation of the MAP kinase pathway 103.

Our results are the first, to our knowledge, to show that ICAM cross-linking promotes skeletal muscle cell proliferation in vitro.

Several studies have examined the cell cycle duration of myoblasts in culture. These studies have shown that the cell cycle for skeletal muscle cell lines (e.g. C2C12) is approximately 12-18 hours 143, 144, while that of primary skeletal muscle cells is 24-32 hours 145, 146. Therefore, we preformed BrdU pulse labeling during various time points throughout the 24 hour growth duration in order to gain additional insight into the kinetics of the ICAM-1- mediated myoblast proliferation. While the prevalence of BrdU incorporation was numerically increased in antibody cross-linked cells compared to controls across the entire 24 hour growth period, the most robust increase was seen from 4-6 hours post cross-linking. This acute response could be due to activation of ICAM-1-mediated signaling cascades that promote the events of proliferation.

Interestingly, activation of ICAM-1 has been shown to activate numerous signaling pathways (i.e. MAPK, Rho kinase, ERK, and PI3K/Akt)

48, 77, 82-84 that are known to promote myoblast proliferation 5, 9, 104, 105, 142. For example, the Raf/ERK pathway has been demonstrated to be required for myoblast proliferation such that inhibition of MKK1/2 blocks the FGF- mediated stimulation of ERK1/2, thereby inhibiting the G1 to S phase

transition of myoblasts 147. Furthermore, others have shown that Akt isoforms are critical for regulating the cell cycle machinery required for myoblast proliferation 148. Future work will be aimed at investigating whether activation of ICAM-1 enhances proliferation of skeletal muscle cells through differential regulation of one or more of these pathways.

It is important however to note that the ICAM-1-mediated myoblast proliferation observed in the present study appears to be dependent on a synergistic effect of serum components in growth medium (e.g. FBS) because we were unable to observe a cross-linking induced increase in proliferation in a number of serum free mediums (Figure 4-3). The mechanism for the observed synergistic effect is currently unknown. One potential mechanism involves serum-derived fibrinogen. Numerous studies have shown that fibrinogen is a potent ligand for ICAM-1 and is capable of activating ICAM-1- mediated signaling both in vivo and in vitro 75, 103, 149-151. Thus, it is possible that fibrinogen is binding to ICAM-1 and acting in concert with antibody cross-linking in order to promote skeletal muscle cell proliferation.

Therefore, future studies aimed at investigating the contribution of fibrinogen to the observed ICAM-1-mediated skeletal muscle cell proliferation are warranted.

The results of the current study show that overexpression of ICAM-1 significantly increases markers of skeletal myotube hypertrophy (Figure 3-

18); whereas activation of ICAM-1 had no effect (Figure 3-19). The

mechanism(s) by which overexpression of ICAM-1 promotes hypertrophy remains to be determined. Given that the observed hypertrophy occurred in the absence of cross-linking, we hypothesize that skeletal muscle cells constitutively express or could be induced to express one or more ligands for

ICAM-1. Such ligands could include β2 integrins (e.g. CD11b/CD18), hematopoietic progenitor cell antigen (CD34), and/or hyaluronan (hyaluronic acid).

One potential mechanism by which overexpression of ICAM-1 could be promoting skeletal muscle hypertrophy is via interaction with endogenous β2 integrins. Classically, it has been thought that β2 integrins are exclusively expressed by cells of the hematopoietic lineage (e.g. neutrophils and macrophages) 17-19 and not by skeletal muscle cells. This is supported by prior experiments in our laboratory which demonstrate that CD18 is not expressed by skeletal muscle cells in-vivo 13. However, more recently, primary muscle satellite cells have been shown to express mRNA for both the alpha and beta chains of the β2 integrin, CD11b/CD18 152. Therefore, although unlikely, it is plausible that β2 integrins could be differentially expressed by skeletal muscle cells and thereby serving as a means by which overexpression of ICAM-1 is promoting skeletal muscle hypertrophy.

Ongoing studies in our laboratory are investigating the expression of CD11a and CD11b by cultured skeletal muscle cells.

Another potential mechanism by which overexpression of ICAM-1 could be promoting skeletal muscle hypertrophy is via interaction with endogenous CD34, a transmembrane glycoprotein capable of binding to

ICAM-1 153, 154. Previous investigators have characterized CD34 as a marker of adult hematopoietic stem cells (HSCs) as well as fibroblasts and endothelial cells 155-159. However, more recently, CD34 has been linked to both the quiescent and activated states of myogenic satellite cells 155, 156.

Functionally, the expression of CD34 was associated with higher regenerative capacities when used in transplantation experiments 156.

However, it has been shown that less than 5% of cultured proliferating myoblasts (i.e. C2C12) express CD34 protein 155. Additionally, in vitro studies have demonstrated that the subpopulations of myoblasts recruited to fuse into myotubes during differentiation are CD34 negative 160. It is currently unknown whether CD34 is interacting with ICAM-1 to facilitate skeletal muscle hypertrophy in our culture system.

Additionally, skeletal muscle cells could be having an autocrine/paracrine effect on ICAM-1 activation through the production and/or secretion of hyaluronan. Hyaluronan is a proteoglycan, composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine that is found in the extracellular matrix as well as cell surface associated 161, 162.

Hyaluronan is the ligand for several cell-surface receptors, including ICAM-1

73, 161-163. Interestingly, hyaluronan has been shown to be produced and

localized to the cell surface of myoblasts and differentiated myotubes as well as secreted in the media 161, 164, 165. Functionally, hyaluronan can promote both the migration 161, 166 as well as fusion/differentiation of myogenic cells

161, 167. The degree to which hyaluronan is expressed by our cultures is currently unknown. If hyaluronan is present, it could be contributing to the observed hypertrophic response either directly, through ligation and activation of ICAM-1, or indirectly, through increased expression of other adhesion molecules (e.g. VCAM-1) 168.

Although not a ligand for ICAM-1, vascular cell adhesion molecule-1

(VCAM-1; CD106) could play a role in augmenting the events of skeletal muscle hypertrophy. Numerous studies have demonstrated that upon ICAM-

1 activation, VCAM-1 expression/recruitment is increased on several cell types 81, 83, 112, 113. In vivo, VCAM-1 has been shown to be constitutively expressed by satellite cells/myoblasts 169 and regenerating myofibers 170, but not by adult muscle fibers 169. In vitro, VCAM-1 has been shown to be constitutively expressed by both myoblasts and myotubes 170. Furthermore, a recent study has shown that treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM-1 expression

171. Interestingly, VLA-4 (α4β1), the receptor for VCAM-1, can not only be expressed by leukocytes, but also by skeletal muscle cells (i.e. myotubes) 169,

170, 172. Therefore, it is plausible that VLA-4-VCAM-1 interactions could play a role in regulating skeletal muscle cell function. Although controversial,

VCAM-1, through interaction with VLA-4, has been shown to promote myogenesis by enhancing the alignment of secondary myoblasts along primary myotubes and/or by enhancing the rate of fusion of various cell types, including muscle cells 170, 171, 173. Furthermore, VCAM-1 on satellite cells is postulated to mediate interactions with VLA-4+ leukocytes that invade the muscle after injury or disease 169. Therefore, based on the cumulative interpretation of prior work, it seems highly likely that VLA-

4/VCAM-1 is expressed and perhaps plays a role in mediating the events associated with ICAM-1-mediated skeletal muscle hypertrophy. Future work will investigate the expression and function of VLA-4/VCAM-1 in our culture system.

4.3 Specific Aim 3

Because activation of ICAM-1 signaling in skeletal muscle cells influenced myogenesis in vitro, we then tested the contribution of ICAM-1 to the hypertrophic response observed in vivo by exposing plantaris muscles of wild type and ICAM-1-/- mice to mechanical loading using the synergist ablation model. We found that ICAM-1 contributes to overload-induced 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 (Figure 3-20).

A limitation of the current study is the use of conventional ICAM-1 knock-out mice. Because ICAM-1 was found to be expressed by skeletal

muscle cells (myofibers and satellite cells/myoblasts), endothelial cells, and leukocytes residing in overloaded muscles, we cannot determine the individual contribution of each ICAM-1 expressing cell type to the hypertrophic response by our comparison of wild type and ICAM-1-/- mice.

Each of the cell types found to express ICAM-1 in overloaded muscles could influence the hypertrophic response via direct and/or indirect mechanisms.

Such mechanisms likely incorporate ICAM-1’s role in mediating β2 integrin- dependent myeloid cell adhesion and its capacity to elicit effector responses in both myeloid cells and other ICAM-1 expressing cell types.

Thus, we are currently unable to substantiate our in-vitro findings that suggest ICAM-1 expression and/or activation could augment skeletal muscle cell function. Preliminary ongoing studies in our laboratory are making additional observations in the overloaded ICAM-1-/- mice in an effort to further characterize the potential mechanisms behind the blunted hypertrophic response. Based on our in-vitro data, we would hypothesize that the reduced hypertrophy in ICAM-1-/- would be associated with decreased satellite cell proliferation, which could ultimately result in decreased hypertrophy 174, 175.

In order to more fully characterize the specific contribution of ICAM-1 expression by skeletal muscle cells to the hypertrophy observed in wild type mice, an in-vivo gene targeting system that allows for tissue-selective (e.g. skeletal muscle specific) gene deletion of ICAM-1 would need to be utilized.

Unfortunately, a “floxed” ICAM-1 mouse has yet to be created, but we believe the results presented herein confirm the need to develop this skeletal muscle specific ICAM-1 knock-out mouse model.

Expression of ICAM-1 by endothelial cells could also contribute to the hypertrophic response to muscle overload by augmenting the accumulation of myeloid cells in overloaded muscles. The expression of ICAM-1 by endothelial cells is required for the trafficking of leukocytes, particularly neutrophils, out of the systemic circulation. Endothelial cell expression of

ICAM-1 mediates firm adhesion and transendothelial migration of neutrophils into skeletal muscle via a CD11b-ICAM-1 dependent mechanism

17, 74, 176, 177. The adhesion and transmigration of monocytes, on the other hand, occurs primarily via a β2 integrin independent mechanism 13, 16.

Consistent with these studies, we found that ICAM-1 expression is required for neutrophil, but not macrophage, accumulation in skeletal muscle after mechanical loading (Figure 3-21). This finding in conjunction with our prior work that demonstrated that β2 integrins contribute to mechanical loading- induced neutrophil accumulation 13, indicate that neutrophil accumulation in skeletal muscle after mechanical loading occurs via the classic β2 integrin-

ICAM-1 paradigm.

Conclusion

The results of the current study demonstrate that ICAM-1 is expressed in and localized to mature skeletal muscle fibers as well as satellite

cells/myoblasts in hypertrophying skeletal muscle following mechanical loading. The expression of ICAM-1 appears to be dependent on events associated with mechanical loading of skeletal muscle cells. Interestingly,

ICAM-1 expression occurred via a β2 integrin independent mechanism.

Functionally, in-vitro studies demonstrated that ICAM-1-mediated signaling promotes the events of proliferation and hypertrophy in skeletal muscle cells.

These results are supported by additional in-vivo experiments utilizing

ICAM-1-/- mice that suggest that ICAM-1 contributes to skeletal muscle hypertrophy. The 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 (Figure 4-4).

Thus, understanding the underlying mechanisms by which myeloid cells contribute to the regulation of muscle size will aid the development of translational strategies (e.g., pharmaceuticals) for maximizing the hypertrophic response to resistance training, particularly for the elderly and patients with inflammatory muscle, cardiovascular, or metabolic disease.

Figure 1-1: Working model for how β2 integrin-ICAM-1

interactions could contribute to the hypertrophic

response to mechanical loading.

Figure 1-2: Working model for how β2 integrin-ICAM-1 interactions could contribute to the hypertrophic response to mechanical loading.

Figure 2-1: Image capture pattern at 10x magnification. The distance between images is depicted as mm.

Figure 3-1: Real-time PCR analysis of ICAM-1 gene expression in wild type plantaris muscle. n = 8 / time point. Mean +/- SEM.

Figure 3-2: A) ICAM-1 immunoblot of wild type plantaris muscles. Molecular weight of ICAM-1 band is 110 kDa. B) Relative abundance of ICAM-1 protein levels in plantaris muscles. *, significantly elevated in overloaded muscles compared to controls (CT). n = 7-8 / time point. Mean +/- SEM.

Figure 3-3: Confocal images of control muscles. A) ICAM-1 (green), B) Merged image of WGA (red) and ICAM-1, C) CD31+ endothelial cells (purple), D) Merged image of ICAM-1 (panel A) and CD31+ endothelial cells (panel C). Approximately 91% of ICAM-1 was found to be co-localized to CD31.

Figure 3-4: Confocal images of 7 and 14 day overloaded muscles. ICAM-1 & CD31: Merged images of ICAM-1 (green) and CD31 (cyan; endothelial cell marker). WGA & DAPI: Merged images of WGA (red; plasma membrane marker) and DAPI (blue).

A 3000 ICAM-1 negative ICAM-1 positive *

)

2 2500



2000

1500

1000

500

Mean Cross Sectional Area ( Area Cross Sectional Mean

0 Control 3 7 14

30 Days of Overload B ICAM-1 negative 25 ICAM-1 positive

2 * 20 ICAM-1 negative = 1959 +- 87 m ICAM-1 positive = 2442 +- 196 m2

Percentage 10

0 500 1000 1500 2000 2500 3000 3500 4000 Cross Sectional Area (m2) Figure 3-5: A) Cross-sectional area of ICAM-1 negative and ICAM-1 positive myofibers in wild type plantaris muscles. Mean +/- SEM. B) Frequency distribution of cross-sectional area of ICAM-1 negative and ICAM-1 positive myofibers in wild type plantaris muscles overloaded for 14 days. *, Significantly higher for ICAM-1 positive compared with ICAM-1 negative myofibers at 14 d of overload.

Figure 3-6: A) Confocal images of 7 d overloaded muscle. Arrow indicates a presumptive satellite cell that is positive for ICAM-1. B) Flow cytometry determination of ICAM-1 expression by satellite cells (CD45negCD31negintegrin alpha 7pos) expressed relative to muscle mass (mg). *, significantly higher for 7 d overloaded muscle compared to controls. Mean +/- SEM.

Figure 3-7: Real-time PCR analysis of ICAM-1 gene expression in plantaris muscles of wild type and CD18-/- mice. n = 8 / time point. Mean +/- SEM.

Figure 3-8: A) ICAM-1 immunoblot of wild type and CD18-/- plantaris muscles. Molecular weight of ICAM-1 band is 110 kDa. B) Relative abundance of protein levels of ICAM-1 in plantaris muscles. *, significantly elevated in overloaded muscles compared to controls (CT). n = 7-8 / time point. Mean +/- SEM.

Figure 3-9: Confocal images of control (CT) and 7 and 14 day overloaded muscles from CD18-/- mice. WGA & DAPI: Merged images of WGA (red; plasma membrane marker) and DAPI (blue; nuclei marker). MERGED: merged images of ICAM-1 and WGA/DAPI.

Figure 3-10: A) Flow cytometry and B) western blot analysis reveals that cultured skeletal muscle cells do not constitutively express ICAM-1, but can be induced to express ICAM-1 with TNF-α treatment. ICAM-1 band is 110 kDa. GAPDH used as a loading control.

Figure 3-11: Protein levels of ICAM-1 and GAPDH. Lanes: 1 = molecular weight standards, 2 = RAW264.7 cells (1 µg; positive control), 3 = non-transfected myoblasts (15 µg), 4 & 5 = myoblasts transfected with empty vector (15 µg / lane), 6 = non-transfected myoblasts (15 µg) treated with TNF-α (10 ng/mL for 24 h), 7 & 8 = myoblasts transfected with ICAM-1 vector (7.5 µg / lane).

Figure 3-12: Overexpression of ICAM-1 does not influence the

ability of skeletal muscle cells to proliferate. n = 6 /

group. Mean +/- SEM.

Figure 3-13: A) Activation of ICAM-1 enhances skeletal

muscle cell proliferation. B) The increase in

cell number after ICAM-1 activation is

specific to the cross-linking protocol. *,

significantly higher in cross-linked ICAM-1+

cells above all control treatments. 1 Ab =

primary antibody only. 1 Iso CT = primary

antibody isotype control (CT). 2 Ab =

secondary antibody only. CT = control

condition. X-L = cross-linking of ICAM-1 via

primary & secondary antibodies. n = 6 /

group. Mean +/- SEM.

Figure 3-14: A) Representative merged images of BrdU (green) incorporation into nuclei (blue) in control cultures or 4-6 hours post cross-linking. B) Cross-linking of ICAM-1 induced a significant increase in BrdU incorporation throughout the 24 hour time course, relative to control cultures. n = 6 / time point. Mean +/- SEM.

Figure 3-15: A) Myogenin immunoblot of skeletal muscle cell differentiation. Molecular weight of myogenin band is ~34 kDa. α- tubulin used as a loading control (~50 kDa). Overexpression of ICAM-1 does not influence the relative abundance of myogenin (B) or fusion index (C) during differentiation. n = 4-6 / group. Mean +/- SEM.

Figure 3-16: A) Myogenin immunoblot in control or activated ICAM-1 cultures during skeletal muscle differentiation. Molecular weight of myogenin band is ~34 kDa. α-tubulin used as a loading control (~50 kDa). Activation of ICAM-1 does not influence the relative abundance of myogenin (B) or fusion index (C) during differentiation. n = 6 / group. Mean +/- SEM.

Figure 3-17: A) Representative merged images of BrdU (green) incorporation into nuclei (blue) in differentiated control cultures or 4-6 hours post cross-linking. B) Cross-linking of ICAM-1 induced a significant increase in BrdU incorporation in day 1 differentiated cultures. *, cross-linked significantly higher than control. n = 6 / group. Mean +/- SEM.

Figure 3-18: A) representative images of myotubes (green

; myosin heavy chain +) from either Empty or ICAM-1+ cultures at day 6 of

differentiation. B – D) Overexpression of

ICAM-1 promotes markers of skeletal

muscle hypertrophy. *, significantly higher

for ICAM-1+ relative to controls (C2C12 &

Empty). #, significantly higher for ICAM-1+

relative to controls (C2C12 & Empty) across

all time points (main effect for cell type). n =

6 / group. Mean +/- SEM.

Figure 3-19: Activation of ICAM-1 does not influence markers of skeletal muscle hypertrophy. n = 6 / group. Mean +/- SEM.

Figure 3-20: ICAM-1 contributes to overload- induced skeletal muscle hypertrophy in vivo. A) Wet muscle mass of plantaris muscles. *, significantly elevated in wild type relative to control and ICAM-1-/- mice. B) Total protein content (µg) in wet plantaris muscles. *, significantly elevated in wild type relative to control and ICAM-1-/- mice. C) Frequency distribution of cross-sectional area of normal myofibers overloaded for 14 d. *, mean cross-sectional area was significantly higher for wild type relative to ICAM-1-/- mice. n = 6 / time point. Mean +/- SEM. 80

Figure 3-21: ICAM-1 expression alters myeloid cell (neutrophil (A) ; and macrophage (B)) accumulation in overloaded skeletal muscle. *, significantly higher for wild type relative to ICAM-1-/- mice. #, significantly higher for wild type relative to ICAM-1-/- mice across all time points (main effect for strain). n = 5-7 / time point. Mean +/- SEM.

Figure 4-1: Real-time PCR for TNF-α gene expression in wild type and CD18-/- mice. β2 integrins contribute to TNF-α

gene expression in overloaded plantaris muscles. #,

-/- significantly higher for wild type relative to CD18 mice across all time points (main effect for strain). n = 8 time point. Mean +/- SEM.

Figure 4-2: ICAM-1 immunoblot of skeletal muscle cells subjected to mechanical loading in vitro.

Figure 4-3: Activation of ICAM-1 does not enhance skeletal

muscle cell proliferation in serum free medium.

Growth response in (A) “basal medium” (DMEM

only) and (B) “insulin medium” (DMEM / F12 +

5 µg/mL insulin). n = 6-8 / group. Mean +/-

SEM.

Figure 4-4: Working model for how β2 integrin-ICAM-1 interactions could contribute to the hypertrophic response after mechanical loading.

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Appendix

Institutional Animal Care and Use Committee Form

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