Integrative Approach to Understanding the Multimodal Effects of Exercise Adaptation

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Alisa D. Blazek, M.A.

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2015

Dissertation Committee:

Dr. Noah L. Weisleder, Advisor

Dr. Timothy E. Hewett

Dr. Sudha Agarwal

Dr. Timothy D. Eubank

Alisa D. Blazek

2015

Abstract

The study of the molecular basis of exercise adaptations in the field of exercise physiology is relatively new, as evidenced by the surge in articles citing molecular techniques over the last decade. This focus on molecular indicators is not surprising given the efforts to improve upon preventative healthcare and reduce healthcare cost burden in our country. The ability to exploit molecular indicators of exercise effectiveness as well as discover novel therapeutic options are clear advantages to studying molecular exercise physiology that could impact healthcare. In the studies described here, we used an integrative approach, building from a molecular basis to mice to human subjects, to develop a more comprehensive understanding of the molecular mechanisms mediating the effects of exercise.

To study the effects of exercise on a physiological systems-wide level, we used microarray technology to characterize global upregulation and downregulation of genes in response to walking exercise in rat cartilage. We found temporal gene expression changes over 15 days of exercise. The networks of genes affected were responsible for directing extracellular matrix; cell metabolism; cytoskeleton; cell signaling, growth, and differentiation; and inflammatory pathways. It was evident from this study that integration of multiple physiological systems occurs in response to an exercise stimulus.

We then aimed to isolate and study selected systems using molecular and physiological techniques. The objective of one particular study was to determine the role ii

of exercise as an integrator of bone and muscle health. One of the genes that was observed during the microarray analysis to be upregulated by exercise by more than 1.5 fold was follistatin-like3 (FSTL3). FSTL3 belongs to the follistatin family of molecules which also includes follistatin (FST). Previous studies showed that FSTL3 is required for exercise driven bone formation. This protein also binds and inhibits myostatin, an inhibitor of muscle growth and strength, thus indicating a role for FSTL3 in hypertrophy and force generation. Using muscle contractility assays and genetic knockout mouse models, it was determined that walking exercise, while sufficient for bone growth, was not a potent enough stimulus for improvements in muscle hypertrophy and force generation.

The follistatin family of proteins have been shown to be involved in cardiac health, thus we aimed to determine the role of follistatin 288 (FST288), a genetic knockout model for follistatin, in the heart with and without exercise and in response to pressure overload induced by transverse aortic constriction (TAC). We found that knockout of the circulating follistatin mediator, FST315, resulted in reduced hypertrophy in response to TAC, indicating that this molecule likely contributes to the generation of pathological hypertrophy in heart failure.

Finally, we aimed to determine the translational potential of our FSTL3 studies in humans. Subjects were exposed to low intensity walking, high intensity walking, or a neuromuscular training program. Bone density, muscular strength, and serum levels of mediators in the follistatin/myostatin system were measured. It was concluded that walking was an insufficient stimulus for increasing FSTL3 to affect bone mass in

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humans, and that FSTL3 was not found to be a suitable biomarker for BMD. However, some gains in muscular strength were observed in previously sedentary patients.

These studies show the importance of integration of multiple indicators

(molecular, clinical, biochemical) in the study of the physiology of exercise, and they advance a growing field of knowledge. Future mechanistic studies of exercise will increase fundamental understanding that could be exploited to improve health by paving a path for biomarker discovery, developing methods for quantitation of exercise effectiveness, and advancing the possibility of personalized exercise prescriptions and novel therapeutics.

Dedication

This document is dedicated to my husband, family and friends for their patience and

understanding.

Acknowledgments

I would like to express my sincere gratitude to Dr. Noah Weisleder, my advisor, for his unwavering support, mentorship, and technical guidance throughout the completion of this work. He unselfishly gave of his time and energy, and I would likely not be at this point without his help and encouragement.

I would like to thank Dr. Tim Hewett, my co-advisor, for believing in me and having a vision for how this work can fit into a broader, translational role. I realize that he has been a powerful advocate for me over the years, and I am truly grateful and humbled by that. I appreciate his positive energy.

Lastly, and certainly not least, I would like to thank Dr. Sudha Agarwal, my co- advisor, for taking a chance on me and finding a way for me to study molecular mechanisms of exercise. I appreciate her hands-on approach in slogging through the day- to-day research with me.

I want to thank Dr. Tim Eubank, my committee member, for serving as an unofficial advisor to me when I first started in my graduate program. He has been a sounding board and mentor to me through the years.

As this work involves multiple projects across multiple labs, it would not have been possible to perform this research without the assistance of various individuals.

I am grateful for the contributions of Dr. Zhaobin Xu, Dr. Karthikeyan

Krishnamurthy, and Kevin McElhanon, members of the Weisleder lab who performed some of the work presented in the heart and recombinant cell treatment sections of this document. The reproducibility of the contractility work would have suffered without the skilled hands of Eric Beck. Dr. Heather Manring provided valuable assistance in breeding and maintaining some of the mouse models used here. In addition, I would like to thank the other members of the Weisleder lab for being a source of assistance in the lab, as well as being a sounding board for technical and graduate school matters, in particular, Jenna Alloush, Brian Paleo, Travis Gurney, Dr. Liubov Gushchina, Anjella

Manoharan, Dr. Sayak Bhattacharya, and Prasanthi Appikatla.

I want to express my gratitude to the members of the Hewett lab: Dr. Stephanie

DiStasi Roewer has been a mentor and incredible role model for me. Dr. Sam Wordeman and Dr. Nienke Willigenburg provided invaluable technical expertise. Lab managers,

Josh Hoffman and Ben Roewer, facilitated this work and were always ready to rescue. I am so grateful to be able to have worked with these five incredibly professional people.

Isac Kunnath, Kari Stammen, and Amy Minnema did an exceptional job of recruitment and blood draws. Rachel Tatarski and Albert Chen performed some of the clinical tests in this study. Dr. Jackie Buell did an amazing job with the bone density scans and providing a great experience for the human subjects by taking time with them to explain the results of tests. Aileen Cudia looked out for me and made things happen when others thought it couldn't be done. Samantha Primmer did much for me behind the scenes that has not gone unnoticed. Chris "Godfather" Nagelli has been like a lab brother to me. I

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am so appreciative of the way he responsibly managed my project when I was not able to be physically present as if it were his own.

Finally, I want to thank the members of the Agarwal lab. Jackie Li did so much to keep the lab running smoothly, especially mouse breeding, upkeep, and exercising.

The mouse experiments presented here could not have happened without her. Jin Nam provided much of the data in the microarray section; I am grateful to be able to carry on his careful research. Although their time in the lab with me was limited, Meera Predhan,

Michelle Williams, and Priyangi Perera provided wonderful assistance and friendship.

Dr. Derrick Knapik helped to me integrate into the lab and taught me much. His assistance was invaluable.

I am appreciative to Ziyue Chen for her assistance with raw data analysis, statistics, and interpretation.

I want to thank the subjects who volunteered their time to participate in the study.

I appreciate their interest, enthusiasm, and desire to make healthy changes in their lives.

The human subjects research in these studies was supported by a Doctoral Student

Research Grant from the American College of Sports Medicine. The molecular studies were supported by the Sigma Xi Grants in Aid of Research and The Ohio State

University Alumni Grants for Graduate Research and Scholarship. Without the support of these funding agencies, the research would not have been possible.

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Vita

1996...... B.A. Zoology, Ohio Wesleyan University

2010...... M.A. Exercise Science, The Ohio State

University, Columbus, OH

1996...... Sales Representative, Laboratory Solutions,

Columbus, OH

1997-1999 ...... Chemist, Princeton Biomolecules

Corporation, Columbus, OH

1999-present ...... Molecular Biologist/Microbiologist, Battelle

Memorial Institute, Columbus, OH

2007-2010 ...... Graduate Teaching Associate, The Ohio

State University, Columbus, OH

2011-present ...... Graduate Research Associate, The Ohio

State University, Columbus, OH

Publications

Nam J, Perera P, Gordon R, Jeong Y, Blazek AD, Kim DG, Tee BC, Sun Z, Eubank TD,

Zhao Y, Lablebecioglu B, Liu S, Litsky A, Weisleder NL, Lee BS, Butterfield T,

Schneyer AL, Agarwal S. Follistatin-like 3 is a mediator of exercise-driven bone

formation and strengthening. Bone 2015 April pii: S8756-3282(15)00151-9.

Blazek AD, Anderson PJ, Brichler JG, Slawinski MK, Rose MT, Kirby TE, Swain CB.

Effects of a Simulated Altitude Device on Endurance Performance and Mucosal

Immunity. JEPonline 2014;17 (6):45-57.

Knapik DM, Perera P, Nam J, Blazek A, Rath B, Leblebicioglu B, Das H, Wu L-C,

Hewett TE, Agarwal SK Jr., Robling AG, Lee B, Agarwal S. Mechanosignaling in Bone

Health, Trauma and Inflammation. Anti-Oxidants and Redox Signaling. Invited Forum

Review Article. 2014 Feb 20;20(6):970-85.

Motawea HKB, Blazek AD, Zirwas MJ, Pleister AP, Ahmed AAE, McConnell BK, and

Chotani MA. Delocalization of Endogenous A-kinase Antagonizes Rap1-Rho-α2C-

Adrenoceptor Signaling in Human Microvascular Smooth Muscle Cells. J Cytol Molecul

Biol. 2014 Jan 10;1(1).

Blazek A, Rutsky J, Osei K, Maiseyeu A, Rajagopalan S. Exercise Mediated Changes in

High-Density Lipoprotein: Impact on Form and Function. Am Heart J. 2013

Sep;166(3):392-400.

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... ix

List of Tables ...... xiii

List of Figures ...... xiv

Chapter 1: Introduction ...... 1

Chapter 2: Cartilage Gene Regulation in Response to Walking Exercise ...... 17

Chapter 3: Exercise as Integrator of Bone and Muscle Health ...... 55

Chapter 4: The Role of Exercise and the Follistatin Mediator in Cardiac Health ...... 95

Chapter 5: Translational Potential of Walking Exercise in Humans ...... 129

Chapter 6: Summary, Significance, and Future Work ...... 167

References ...... 177

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List of Tables

Table 1. Extracellular Matrix Biosynthesis and Metabolism Genes Regulated in Cluster I

...... 37

Table 2. Inhibitors of ECM Degradation Genes Regulated in Cluster I ...... 38

Table 3. ECM Remodeling Enzymes Regulated in Cluster I ...... 38

Table 4. Biosynthesis of Intermediate Metabolite Genes Regulated in Cluster II ...... 39

Table 5. Cell Signaling, Growth, and Differentiation Genes Regulated in Cluster III ..... 40

Table 6. Cell Cycle Genes Regulated in Cluster III...... 41

Table 7. Cytoskeleton, Ion Channel, and Adhesion Molecule Genes Regulated in Cluster

IV ...... 43

Table 8. Inflammation Related Genes Regulated in Cluster V ...... 44

Table 9. Subject Characteristics ...... 141

Table 10. Average Bone Mineral Density (g/cm2) ...... 143

Table 11. Serum FSTL3 Concentration (pg/mL) ...... 152

Table 12. Serum MSTN Concentration (ng/mL) ...... 155

Table 13. Serum FST Concentration (ng/mL) ...... 157

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List of Figures

Figure 1. FSTL3 Expression in Response to Walking Exercise in Various Tissues ...... 7

Figure 2. Structure of the Follistatin Family Members Relevant to these Studies...... 8

Figure 3. Gene Expression from Rat Cartilage Samples ...... 24

Figure 4. Clusters showing Trends in Temporal Gene Expression ...... 25

Figure 5. Molecular Networks Generated by IPA from the Skeletal and Muscular

Development and Function Genes in Clusters I and IV ...... 28

Figure 6. Molecular Networks Generated by IPA from the Cell Cycle Genes in Clusters II and VI...... 29

Figure 7. Molecular Networks Generated by IPA from the Carbohydrate Metabolism

Genes in Clusters III and VII...... 30

Figure 8. Molecular Networks Generated by IPA from the Cellular Development Genes in Cluster III...... 31

Figure 9. Molecular Networks Generated by IPA from the Immune Cell Trafficking

Genes in Cluster V...... 32

Figure 10. Molecular Networks Generated by IPA from the Connective Tissue

Development Genes in Cluster VI...... 33

Figure 11. Molecular Networks Generated by IPA from the Skeletal and Muscular

Disorder Genes in Cluster VIII...... 34

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Figure 12. Molecular Networks Generated by IPA from the Cellular Growth and

Proliferation Genes in Cluster IX...... 35

Figure 13. Molecular Networks Generated by IPA from the Inflammatory Response

Genes in Cluster IX...... 36

Figure 14. Specific Force (N/cm2) of EDL and SOL Muscles from WT Male Mice in

Response to Stimulation Frequencies from 1-150 hz...... 65

Figure 15. Specific Force (N/cm2) of EDL and SOL Muscles from FSTL3 KO Male Mice in Response to Stimulation Frequencies from 1-150 hz...... 66

Figure 16. Specific Force (N/cm2) of EDL and SOL Muscles from WT Female Mice in

Response to Stimulation Frequencies from 1-150 hz...... 67

Figure 17. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FSTL3 KO

Female Mice in Response to Stimulation Frequencies from 1-150 hz...... 68

Figure 18. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FST288 Male

Mice in Response to Stimulation Frequencies from 1-150 hz...... 69

Figure 19. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FST288

Female Mice in Response to Stimulation Frequencies from 1-150 hz...... 70

Figure 20. Specific Force (N/cm2) of EDL and SOL Muscles from FST288, FSTL3 KO, and DKO Male Mice in Response to Stimulation Frequencies from 1-150 hz...... 72

Figure 21. Specific Force (N/cm2) of EDL and SOL Muscles from FST288, FSTL3 KO, and DKO Female Mice in Response to Stimulation Frequencies from 1-150 hz...... 73

Figure 22. Specific Force (N/cm2) of EDL and SOL Muscles from WT and DKO Mice in

Response to Stimulation Frequencies from 1-150 hz...... 74

Figure 23. Muscle Weight to Body Weight Ratios from Male and Female FSTL3 KO

Mice Compared to WT Mice...... 75

Figure 24. Representative Graph of Comparison of Percent Fatigue between FSTL3 KO and WT Muscles...... 77

Figure 25. Experimental Design for Transfection of Mouse Myoblast C2C12 Cells with

Fstl3...... 79

Figure 26. Results of the Transfection of Mouse Myoblast C2C12 Cells with Fstl3

Experiment...... 80

Figure 27. Experimental Design for the Addition of Recombinant FSTL3 to Mouse

Myoblasts and Myotubes Experiment...... 81

Figure 28. Confirmation of Myotube Development in Cellular Extracts from the

Recombinant FSTL3 Treatment Experiment...... 82

Figure 29. Myotube Size in Response to Recombinant FSTL3 Treatment...... 83

Figure 30. Heart Weight to Body Weight (HW/BW) and Heart Weight to Tibia Length

(HW/TL) Ratios for FSTL3 KO Hearts Compared to WT Hearts...... 107

Figure 31. Ejection Fraction and Fractional Shortening Measurements for FSTL3 KO

Hearts Compared to WT Hearts...... 108

Figure 32. Left Ventricular Internal Dimension Measurements for FSTL3 KO Hearts

Compared to WT Hearts...... 109

Figure 33. Left Ventricular Internal Dimension Measurements for DKO Hearts

Compared to WT Hearts...... 110

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Figure 34. Heart Weight to Body Weight (HW/BW) and Heart Weight to Tibia Length

(HW/TL) Ratios for FST288 Hearts Compared to WT Hearts...... 111

Figure 35. FST288 Hearts show Potential Differential Remodeling in Response to

Exercise Training...... 113

Figure 36. FST288 Hearts show no Changes in Function in Response to Exercise

Training...... 114

Figure 37. FST288 Hearts Display Blunted Hypertrophic Response after TAC...... 116

Figure 38. Structural Measurements in Response to TAC...... 117

Figure 39. Functional Measurements in Response to TAC...... 118

Figure 40. Examination of Hypertrophic Signaling Pathways in Hearts Subjected to TAC.

...... 119

Figure 41. Examination of TGF- Signaling Pathways in Hearts Subjected to TAC. ... 121

Figure 42. Average Subject Characteristics...... 142

Figure 43. Total Bone Mineral Density Results...... 144

Figure 44. Bone Mineral Density Site Results...... 145

Figure 45. Correlation between FSTL3 Serum Concentration and BMD...... 146

Figure 46. Hip Strength Testing Results...... 149

Figure 47. Biodex Testing Results at 60 Degrees per Second...... 150

Figure 48. Biodex Testing Results at 300 Degrees per Second...... 151

Figure 49. Comparison of Serum FSTL3 Concentrations between and within Groups. 153

Figure 50. Comparison of Serum MSTN Concentrations between and within Groups. 156

Figure 51. Comparison of Serum FST Concentrations between and within Groups...... 158

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Chapter 1: Introduction

Exercise is defined as: “planned, structured, and repetitive, with the intent of improving or maintaining one or more facets of physical fitness or function.”1 As a general health modifier, exercise has been used successfully to counter age-related osteopenia and sarcopenia.2,3 While it is well known that exercise is important for fitness and health maintenance, recent reviews of exercise therapy for bone and muscle health have overwhelmingly concluded that exercise is recommended to decrease pain and increase physical function in a multitude of musculoskeletal disorders.4, 5, 6, 7, 8

Interestingly, these reviews acknowledge the significant gap in the understanding of how exercise improves clinical outcomes. Closing this gap by increasing knowledge of the mechanistic basis for the clinical effectiveness of exercise would enable the most suitable applications of exercise for physical therapy to treat musculoskeletal disorders as well as for personal training to maintain health. The National Institutes of Health recently convened a workshop to identify knowledge gaps and strategies for improving progress in the field of “cellular and molecular mechanisms of physical activity health benefits.”9

The importance of such an undertaking is obvious, as the discovery of molecular indicators could make an impact in estimating the effectiveness of exercise, reducing healthcare costs and improving preventative healthcare. A personalized approach to exercise prescription appears to be the future of medicine; however, much work needs to be done before this vision can become a reality.10, 11, 12 To begin making strides in this 1

area of research of the cellular and molecular mechanisms of exercise, we examined potential mechanisms on the organismal as well as specific systems level, including the musculoskeletal system and heart, using cell and animal models. We also used human subjects to determine the translational potential of our work. Our studies also targeted several disease states known to be modified by exercise, including osteoarthritis, disuse atrophy, and heart failure. Such an approach provides a useful roadmap for future research in this area, as it was evident from our studies that integration of multiple physiological systems occurs in response to an exercise stimulus.

We first studied the effects of exercise on a physiological systems-wide level by using microarray analysis to characterize global upregulation and downregulation of genes in response to walking exercise in rat cartilage. Cartilage is an important tissue to study because of the healthcare problem of osteoarthritis (OA), which is a degeneration of the cartilage covering the joints. OA of the hip and knee is one of the leading causes of global disability,13 and increasing age and obesity of the U.S. population is resulting in higher healthcare costs for OA treatment.14, 15

Structure and Function of Cartilage

Articular cartilage is composed of extracellular matrix (ECM) that is synthesized by the primary cell type, the chondrocyte.16 The ECM contains the main constituents of articular cartilage, type II collagen and the proteoglycan aggrecan,17 as well as other collagens, non-collagenous glycoproteins, hyaluronan, other proteoglycans,18 chondroitin sulfate,19 and proteins such as versican, biglycan, elastin, decorin, syndecan, cartilage intermediate layer protein (CILP), and cartilage oligomeric protein (COMP).20, 21 The

ECM is dynamic, accommodating growth factors and cytokines to regulate cell turnover, proliferation, and differentiation.18 Similarly, the homeostasis of the ECM is maintained by remodeling, degradation, and turnover regulated by the chondrocytes.18 In healthy cartilage, chondrocytes are very metabolically active cells that continually synthesize matrix, while secreting factors that minimize the activity of matrix degrading enzymes, such as matrix metalloproteinases.22

During exercise, stimulation of healthy cartilage by mechanical forces provides signals to the cells and ECM, which can respond to their mechanical environment.21

Integrins on the surface of the chondrocytes are transmembrane receptors that connect with the cytoskeleton through their cytoplasmic domain.17 These molecules perceive mechanical signals, and serve as a communication link between the chondrocytes and the

ECM,17 making the cartilage a mechanoresponsive tissue.21 Other transmembrane molecules on the chondrocyte include the annexins, which enable chondrocyte binding to

Type II collagen, and the hyaluronan receptor CD44, which is needed for cartilage homeostasis.17 In addition to chondrocytes, the ECM contributes to the mechanosensitive properties of cartilage. These properties are defined by collagen, which provides tensile strength, and aggregan, which provides elasticity.18

Cartilage is an avascular and anervous tissue,23 making healing from injury difficult. Injury, high mechanical stress, or systemic inflammation as a result of various insults can lead to the development of osteoarthritis (OA).24 In OA, quiescent chondrocytes begin to proliferate, form clusters, and increase production of ECM- degrading enzymes.24 Infiltration of inflammatory cells and soluble mediators into the

joint is common in OA, compromising the homeostasis of the chondrocyte, and leading to matrix and cartilage breakdown. Eventually, the chondrocytes undergo apoptosis, leading to loss of the cartilage.25

Significance and Pathology of Osteoarthritis

Although OA was once believed to be a disease involving mechanical degradation of the cartilage, it is now understood that its onset and progression is multifactorial, with potential involvement of the cartilage, subchondral bone, synovium, and inflammatory cells.26 Three proteolytic enzymes are most associated with the degradation of cartilage in OA: A disintigrin and metalloproteinase with thrombospondin motif (ADAMTS) 4 and

5, which degrade aggrecan, and matrix metalloproteinase 13 (MMP13), which targets collagen II.27, 25 However, the pathology of OA is complicated, and other processes may also lead to cartilage degeneration. These processes include hypoxia-inducible transcription factor (HIF)2α induction of inflammation, reduction in cellular autophagy that normally maintains a balance during periods of cell stress, and alterations in signaling pathways that are normally tightly regulated, such as Wnt/β-catenin.27, 25

Besides degradation of the cartilage, other pathologies of the disease can include subchondral bone remodeling, osteophyte formation, and participation of the synovium in inflammatory processes.27 Recent MRI studies have indicated the possible early involvement of the subchondral bone, which has been shown to correspond to clinical symptoms of pain due to its highly innervated structure.28 Other studies suggest that subchondral bone remodeling could result from increased loading due to the loss of the cartilage.26 The innate immune system has been found to be active in OA, including an

abnormally active complement cascade in affected joints, as well as activation of toll-like receptors on the surface of chondrocytes by ECM components.26 Systemic inflammation also has been observed, but studies are conflicting on this point.26 It is believed that adipokines released from fat tissue could play a role in increasing risk in OA in non- weight bearing joints.29 Finally, synoviocyte proliferation and increased vascularity are commonly observed in OA.26

Unfortunately, diagnosis of OA is made only after the onset of symptoms, which in most cases represents an advanced and irreversible stage of disease. Diagnosis is confirmed by radiography to observe narrowed width of the joint space, formation of osteophytes, and subchondal abnormalities,26 although MRI is a more sensitive diagnostic method. Multiple blood biomarkers for diagnosis have been proposed, but none have been sufficiently validated to put into clinical practice.26 Use of blood biomarkers could be advantageous for diagnosis, especially when coupled with other methods.

We determined through our microarray analysis that exercise has effects on healthy cartilage that could decrease risk for OA. In addition, our microarray analyses also uncovered a novel circulating, mechanosensitive protein, follistatin-like 3 (FSTL3), which mediates exercise-driven bone formation30 and may provide insights into how exercise modifies musculoskeletal health. Microarray analysis revealed that FSTL3 was significantly upregulated in cartilage, bone, and serum in walking rats.30 Quantitative real-time PCR analysis of multiple tissues confirmed that FSTL3 was significantly upregulated in bone and cartilage as well as in muscle (Figure 1) after two days of walking exercise. The fact that FSTL3 is a circulating mediator and is upregulated in

tissues important for exercise led us to suspect that FSTL3 could play a role in mediating bone and muscle communication and adaptation in response to exercise. We therefore aimed to isolate and study a selected system, in this case, the musculoskeletal system, using molecular techniques. The objective of this particular study was to determine the role of exercise as an integrator of bone and muscle health through the FSTL3 mediator.

The Follistatin Family

The follistatin family members consist of proteins containing follistatin domains, conserved sequences of ten cysteine residues, which also bind and antagonize the transforming growth factor (TGF)-β family molecules, activin and myostatin (MSTN).31,

32, 33, 34 Although other proteins may contain follistatin domains as well, the follistatin family members are unique in their abilities to bind activins.35 These family members differ in their protein structures (Figure 2). The follistatin (FST) protein isoforms, which all contain three FST domains and a heparin binding site, begin as a 344 amino acid precursor.36 Cleavage of the signal peptide results in the mature FST315 form. Further cleavage at the C-terminus of FST315 results in FST303. Alternative splicing of the primary transcript results in FST317, which is then cleaved to produce the mature

FST288 form.37 While the mature FST288 can bind cell-surface heparin-sulfated proteoglycans, the acidic tail of FST315 prevents this interaction; thus, FST315 is largely a circulating protein.38 The function of FST303 has not been documented, and has not been studied here.

The follistatin like (FSTL) proteins are related family members that contain only two follistatin domains. FSTL3 does not contain a heparin binding site, and like FST315,

is a circulating protein.38, 35 Although FST and FSTL3 are structurally similar, the lack of one follistatin domain and heparin binding domain in FSTL3 lends unique functional properties to these two related proteins that will be described further later.39, 40

FSTL1 does not bind to activin and contains two EF-hand calcium binding sites.41

Other FSTL molecules are not well studied, and are not covered in this review in detail.

Figure 1. FSTL3 Expression in Response to Walking Exercise in Various Tissues

The data represent real-time quantitative PCR analysis of mRNA extracted from n=3 samples per group at each time point. *p<0.05

Figure 2. Structure of the Follistatin Family Members Relevant to these Studies.

FSTL3 is similar in structure to FST, but lacks a follistatin domain. The FST isoforms are formed from proteolytic cleavage (FST315, FST303) or alternative splicing (FST288) of the primary FST344 protein.

Follistatin-like 3 is a Binding Partner and Inhibitor of Myostatin in the Serum

FSTL3 is the circulating binding partner of MSTN, an inhibitor of muscle hypertrophy, and may be responsible for MSTN inhibition by preventing it from binding to its receptor.33, 32 Since MSTN inhibits myogenesis, the binding and inhibition of

MSTN by FSTL3 potentially could promote muscle hypertrophy. An experiment using a monoclonal antibody to isolate MSTN complexes from serum and then subsequent analyses of the complexes by mass spectrometry indicated that FSTL3 and the myostatin propetide are the ONLY binding partners of MSTN in the mouse and human; FST was not found to bind MSTN in the serum in vivo.34 Few studies detail the regulation of

FSTL3. TGF-β, activin A, prostaglandin E2 (PGE2), and the protein kinase C activator

TPA (12-O-Tetradecanoylphorbol-13-acetate) have been shown to increase the transcription of Fstl3 through Smad proteins.42

FST and its Differences and Similarities to FSTL3

FST, another member of the follistatin family which is related to FSTL3, also is present in many types of cells and has been shown to be upregulated in human serum by three hours of cycling exercise, and in mouse plasma and liver after a one hour bout of swimming.43 Follistatin was first discovered by its ability to suppress secretion of follicle stimulating hormone from anterior pituitary cell monolayer cultures by neutralizing endogenous activin.44, 45 Follistatin mRNA has been detected in many tissues in the rat

(ovary, testes, cerebral cortex, pituitary, adrenal, thymus, pancreas, gut, kidney, heart, uterus, skeletal muscle and lung).46 Global gene deletion of FST in mice results in skeletal defects, weakened musculature and death of neonates shortly after birth.47, 48

These mice fail to breathe and die within hours after birth with multiple abnormalities of the diaphragm and intercostal muscles, skin, hard palate and thirteenth pair of ribs, and whiskers and teeth.47 Gene deletion of FST315 (and thus FST303) is not lethal and results in mice with a normal phenotype.49, 47 Follistatin counterbalancing of TGF-β signaling is important for normal tissue development and function.50, 51, 52, 53, 54 FST315 appears to be secreted faster and is the major isoform present in the circulation due to its lack of ability to bind cell surfaces due to its acidic tail, whereas FST288 can bind to cell surface heparin-sulfated proteoglycans.55, 56 The role of FST in mechano-induced bone growth has not been determined; however, evidence suggests a likely role of FST in the regulation of bone and muscle growth, and it is possible that FST and FSTL3 share

functional similarities in regulating bone and muscle growth and strengthening. Similar to FSTL3, FST can neutralize MSTN, as shown by in vitro competitive binding assays.57

FST is known to regulate BMP-7 to promote embryonic muscle growth.58

Although FST and FSTL3 are structurally similar, the lack of a heparin binding domain and one less follistatin domain in FSTL3 lends unique functional properties to these two related proteins.39, 40 For example, both molecules bind activin, but FST is

100X more potent in this regard than FSTL3.59 FSTL3 and FST regulate their own expression through a negative feedback loop with activin: An increase in FSTL3 or FST results in a decrease in activin, as both FST and FSTL3 bind to and inhibit activin; however, activin signaling increases the expression of FST and FSTL3.60 Although FST and FSTL3 are expressed ubiquitously, peak distribution differs, with FSTL3 predominanting in the placenta, testis, and cardiovascular tissue, and FST predominating in the ovary and pituitary. Only FSTL3 was identified as having the potential for nuclear localization.61 Relative expression of these two mediators in mouse fetal tissues and in several human cell lines also differs.61 FST288 mice appear to weigh less than their WT counterparts, while no differences from WT were noted for Fstl3-/- mice. Knockout of the different isoforms appear to impact glucose and fat homeostasis.62, 31, 63 The potential for both FST and FSTL3 to bind and inhibit the actions of MSTN suggests a possible role for these molecules in muscle growth and strength.

Structure and Function of MSTN

The MSTN precursor protein contains 376 amino acids and a signal sequence, an

N-terminal propeptide domain, and a C-terminal domain.64 This precursor, as well as the

mature MSTN, forms disulfide-linked dimers. The MSTN dimer remains latent when complexed with its propeptide and until proteolytic processing releases the mature

MSTN, activating it, and allowing it to bind to its receptors, the activin Type II receptors

(ActRIIB).65 The Type I receptor (Alk-4/5) is then recruited and activated by the Type II receptor, and Smad (mothers against decapentaplegic homolog) 2/3 proteins are activated and form a complex with Smad4.66 Smads regulate myogenic gene expression and can inhibit MyoD, a transcription factor that increases myoblast proliferation. In fact, MSTN affects muscle growth through several mechanisms. For example, MSTN also inhibits the protein kinase B (AKT)/insulin-like growth factor (IGF-1) pathway, resulting in inhibited protein synthesis (inhibits mammalian target of rapamycin, mTOR, pathways)67 and increased ubiquitin ligase pathways (through FoxO1 transcription factors). Further, treatment with recombinant MSTN showed that the MEK1/Erk1/2 MAPK pathway was activated in C2C12 myoblasts through Ras Raf to control cell cycle and negatively regulate muscle cell differentiation.68 Reduced levels of myosin heavy chain (MyHC), myogenin (another transcription factor involved in muscle development), and MyoD were observed in response to MSTN treatment. In addition, these effects were shown to be mediated through the ActRIIB using siRNA knockdown of the receptor mRNA. In a separate study, C2C12 myoblasts were incubated with increasing levels of MSTN, and fluorescence-activated cell sorting showed that these myoblasts were prevented from progressing from G1 to S phase of the cell cycle.69 Western analyses showed an increase in p21 (a cyclin dependent kinase inhibitor) expression and a decrease in Cdk2 (cyclin- dependent kinase 2) protein, leading to a decrease in phosphorylated Rb protein

(phosphorylated retinoblastoma protein results in cell cycle progression). The end result of MSTN signaling is increased muscle atrophy and decreased muscle hypertrophy.70

The Role of MSTN in Muscle Development

MSTN is expressed from early development (somite myotome compartment) and in the myogenic lineage through adulthood.65 A pool of quiescent muscle cells, the satellite cells, reside between the basal lamina and sarcolemma of the myofibers.71 When activated by an injury or growth stimulus, satellite cells re-enter the cell cycle, divide, differentiate, and fuse into myofibers. Studies are mixed on the response of satellite cells due to MSTN inhibition. Some studies report an increase in satellite cell number and activity in response to MSTN inhibition, while others show no change.72 Supporting the belief that MSTN signals directly to the myofiber versus the satellite cells is the observation that muscle hypertrophy can occur with MSTN inhibition in the absence of satellite cell activity.73

Effects of MSTN Ablation and Inhibition

Spontaneous MSTN mutants display hypermuscular phenotypes in cattle74 and in dogs.75 MSTN ablation results in both hyperplasia and hypertrophy of muscle fibers, although increased muscle mass has been observed to be caused by hyperplasia without hypertrophy or hypertrophy without hyperplasia; these differences may be the result of species differences and degree of MSTN inhibition.76 Transgenic mice generated to express the MSTN propeptide, FST, or a dominant-negative form of ActRIIB specifically in skeletal muscle displayed dramatic increases in muscle mass due to hypertrophy and hyperplasia of muscle fibers.65

Effects of Exercise on MSTN Expression

MSTN expression has been shown to decrease in muscle and in plasma in response to exercise,77, 78 but contradictory observations have been reported. For example, it has been shown that MSTN can also increase in serum and muscle in response to exercise, as shown in a study involving 12 weeks of heavy resistance training in humans.79 However, there was a concomitant increase in FSTL3 as well as a downregulation in the activin receptor that negated the potentially muscle reducing effects. It appeared that MSTN was increasing with muscle damaging, eccentric exercises in this particular study. Hard training also has been shown to increase serum cortisol, which upregulates the glucocorticoid receptor and signaling, leading to an increase in MSTN, which has a glucocorticoid response element in its promoter.

However, other studies report no change in MSTN expression in response to eccentric exercise80, 81, 82 or even a decrease.83, 84,85 The degree of MSTN reduction with exercise may depend on exercise type, intensity, and training status, and sampling time post exercise can also impact MSTN concentration levels.70

Regulators of MSTN Action

Negative regulators of the MSTN protein include SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), as well as FSTL3 and FST.86, 65 SMURF1 tags SMAD 2/3 for degradation, while FSTL3 and FST bind to MSTN and prevent it from binding to its receptor. Another MSTN inhibitory protein, growth and differentiation–associated serum protein-1 (GASP-1), is a secreted glycoprotein that binds both mature MSTN and the propeptide. 87 Overexpression of GASP-1 results in increased myofiber hypertrophy but

not hyperplasia or change in fiber types, suggesting that GASP-1 does not affect MSTN during prenatal development.87 MSTN binding proteins in the serum include FSTL3

(mature form only) and GASP-1 (mature and propeptide forms), and in the muscle include hSGT (human small glutamine-rich tetratricopeptide repeat-containing protein), titin cap, and FST.75, 88 hSGT may function as a chaperone for the full length MSTN protein, and both hSGT and titin cap may mediate MSTN secretion and activation in muscle.89 MSTN gene expression is negatively regulated by Smad 765 and positively regulated by FoxO.90

Molecular and Physiological Effects of MSTN Signaling

MSTN inhibits skeletal muscle growth through inhibition of myoblast proliferation and differentiation.91, 69, 92 Muscle precursor cells become committed to the myogenic lineage by the muscle specific transcription factors, myoD and myf5.92 These cells then progress to myotubes due to the influence of myogenin and muscle regulatory factor 4.92, 93 Overexpression of MSTN in C2C12 myoblasts has been shown to reduce myoD and myogenin expression, and therefore, reduces myoblast progression through the cell cycle.

In the adult, muscle satellite cells are quiescent until activated to enter the cell cycle and express myogenic factors. These satellite cells self-renew, and their numbers remain constant. MSTN inhibits the progression of myoblasts from G1 to S phase of the cell cycle, and serum concentrations of MSTN have been shown to be increased in muscle wasting disorders.94 Increased numbers of satellite cells have been observed with

MSTN null mice. MSTN null cells remain in the cell cycle longer, express MyoD longer,

and delay expression of the differentiation specific myogenin.94 MSTN further decreases muscle hypertrophy by decreasing protein synthesis,95 and increasing expression of protein degradation genes.96 MSTN also affects the cell cycle; p21 is a cell cycle inhibitor that is a target of MSTN signaling.69

Comprehensive Understanding of the Multimodal Effects of Exercise Requires an

Integrative Analysis Approach

It is clear that MSTN, FSTL3, and FST are factors that could potentially affect musculoskeletal health. The relationships between these molecules and their effects on bone and muscle are somewhat known, but also require continued study to determine mechanisms of action and clinical effects. While continued mechanistic research on the musculoskeletal system is important, we also wanted to study other systems as well as apply the results of the musculoskeletal work to a human population. Thus, we next aimed to determine the role of follistatin in the heart with and without exercise and in response to pressure overload induced by transverse aortic constriction (TAC). In addition to its roles in the musculoskeletal system, the follistatin family of proteins in particular also have been shown to be involved in cardiac health. Finally, we aimed to determine the translational potential of our musculoskeletal studies in humans.

The integration of all of these studies represent a body of work that will contribute to the continued expansion of an emergent field of knowledge, that of the molecular and cellular basis of exercise adaptation. These studies show the importance of the integration of multiple investigative methods (molecular, clinical, biochemical) in the study of the physiology of exercise in both health and disease. Future mechanistic

studies of exercise will increase fundamental understanding that could be exploited to improve health by paving a path for biomarker discovery, developing methods for quantitation of exercise effectiveness, and advancing the possibility of personalized exercise prescriptions and novel therapeutics.

Chapter 2: Cartilage Gene Regulation in Response to Walking Exercise

Introduction

It is well known that exercise induces a multitude of physiological changes in the body. Recent studies have shown that both resistance and aerobic exercise are potent signals for alterations in global and muscle gene expression.97, 98, 99, 100 However, studies on the gene altering effects of exercise on healthy cartilage are lacking. Many studies report that exercise is protective to the cartilage,101, 102, 103, 104, 105, 106, 107 preventing or delaying pathological changes that can result in osteoarthritis (OA).108 However, opposing observations also have been reported,109, 110, 105 and it is possible that cartilage response to exercise depends on the health status of the tissue that exists before the exercise is administered,111, 112 and intensity of exercise,113, 114 among other potential factors, especially to those at risk of OA. Therefore, our aim in this study was to determine the genes within healthy cartilage that are most highly regulated in response to exercise.

Previous gene expression studies analyzed temporal gene expression in the heart, skeletal muscle, and brain in response to exercise. Markers of mitochondrial adaptation to aerobic exercise were found within skeletal muscle after one week of repeated exercise sessions in both humans115 and zebrafish,116 and global muscle changes were found within as little as two hours after endurance exercise in Alaskan sled dogs.117 Similarly, muscle appears to begin to adapt to resistance exercise within hours of a session.118, 119, 17

120 Other studies have characterized temporal gene expression patterns in the nervous system in response to exercise,121, 122, 123 and in the heart.124 While the necessity of characterization of the gene changes in working skeletal muscles and in the brain controlling the muscles in response to exercise appears obvious, a thorough examination of cartilage response to exercise has been overlooked. However, study of healthy cartilage tissue is certainly warranted, as it provides essential cushioning for the joints, reducing friction and allowing fluid movement of the bones at the joints. Degeneration of articular cartilage results in OA pathology and progression, leading to significant disability and reduced quality of life.

Several studies have examined gene expression changes in OA cartilage with and without exercise. In one study, biopsies of the articular cartilage were obtained from healthy donors as well as patients with diagnosed OA. RNA analyzed from the biopsies indicated that new candidate genes not described at that time and were associated with bone and collagen were upregulated in the OA donor cartilage.125 In another study, arthritis was induced in rat knees. Genes controlling inflammatory mediators, receptors, and proteases showed temporal regulation during the progression of cartilage destruction.126 Treadmill walking was shown to suppress proinflammatory genes after arthritis induction in rats, but the extent of cartilage damage at the initiation of exercise was an important determinant for exercise effectiveness.127 Finally, gene expression patterns from inflamed and normal areas of arthritic cartilage were examined.

Differential expression between these two areas was most closely related to pathways controlling inflammation, cartilage metabolism, Wnt signaling, and angiogenesis.128

Low intensity exercise has been shown to suppress inflammatory genes and upregulate anabolic repair mechanisms during the progression of low grade OA.127 In fact, multiple studies indicate that exercise should be recommended for the management of OA.129, 130, 131 In particular, an eight week lower body strength training program was shown to reduce knee adduction moment (which is related to joint space width and severity of knee OA) in early OA.132 A home based exercise program using resistance bands was shown to reduce OA knee pain.133 A randomized, controlled trial comparing both aerobic and resistance exercise programs showed that both exercise types improved measures of disability, physical performance, and pain.134 An eight week walking program improved functional status in OA.135 In general, reviews show that exercise is beneficial for the treatment of OA.136, 137

Mechanistic studies to explain how exercise prevents the onset and progression of

OA in healthy cartilage are limited, but recent genome wide association studies (GWAS) have identified potential OA associated genes.138, 139 Similarly, genetic polymorphisms associated with OA also have been described.140, 141, 142 Certain genetic variants may modify susceptibility to OA in response to exercise,143, 144 indicating that genetic factors can influence interactions between exercise effects and the development and progression of OA. Therefore, the aim of this work was to investigate sustained changes in gene expression profiles in healthy rat articular cartilage during the beginning of a low intensity exercise program. Determination of gene expression changes in healthy cartilage in response to exercise could provide clues to mechanisms of OA onset and progression at the genetic level.

No gene expression studies on the effects of exercise on healthy cartilage exist, to our knowledge. In this study, we aimed to conduct an investigation of gene expression changes in healthy, rat articular cartilage in response to low intensity, walking exercise

(EX). A temporal transcriptome analysis was conducted on the knee cartilage of rats after 2, 5, or 15 days of EX and compared to the transcriptome of sham control cartilage.

Ingenuity Pathways Analysis (IPA) was used to determine relationships between genes during the early progression of an EX program. While studies of gene expression in cartilage in response to non-impact, swimming EX have been performed previously,145 to our knowledge, our study is the first to determine temporal gene regulation and molecular networks in healthy cartilage in response to low intensity, cyclical loading exercise.

Determining such changes in gene expression can serve as a framework to characterize healthy versus pathological tissue states and will allow us to understand basic mechanisms of exercise that can give insight into manipulation of exercise protocols to optimize therapeutic value in both health and in diseases such as OA.

Materials and Methods

Exercise regimens

All experiments were conducted following approval from the Institutional Animal

Care and Use Committee and the Institutional Review Board at The Ohio State

University (OSU). Sprague Dawley rats (n=3/group, 12-14 wk old females, Harlan Labs,

IN) were exercised by treadmill walking at 12 m/min for 45 min daily for 2, 5, or 15 days. All animals, including non-exercised controls, were allowed normal cage activity

during the remaining time.126 Rats were sacrificed 2 hours after the last exercise regimen, and femurs were harvested and snap-frozen in liquid nitrogen for microarray analysis.

Cartilage processing

Superficial articular cartilage on the patellar and condylar surfaces of the distal ends of each of the frozen femurs was removed (10–15 mg/femur) and pulverized into 1

µm fragments in a Mikrodismembrator S (Sartorious, France) at 2500 rpm for 30 seconds.127 RNA was extracted using Trizol reagent (Invitrogen, CA), and RNA quality was verified by analysis in a 2100 Bioanalyzer (Agilent, CA).146 The Whole Transcript

(WT) cDNA Synthesis and Amplification Kit and WT Terminal Labeling Kit

(Affymetrix, CA) were used for cDNA synthesis and labeling from each 300 ng RNA template. Labeled samples were hybridized to Affymetrix GeneChip Rat Gene 1.0 ST

Array and scanned at the Microarray Shared Resource Facility at the OSU

Comprehensive Cancer Center.

Microarray and statistical analysis

Partek Genomic Suite version 6.4 (Partek Inc., MO) software was used to analyze intensity scans from three biologically independent arrays per exercise protocol in order to determine changes in gene expression. ANOVA was used to calculate differences among exercise protocols, and only significantly regulated transcripts (p<0.05) were considered for further analyses. Variations among the samples from each exercise protocol were examined by principal component analysis (PCA), and were subjected to both hierarchical and partition clustering by Partek Genomic Suite. PCA is a method of statistical analysis that fits the data to an ellipsoid and graphically depicts the variance in

large data sets. Heirarchical clustering finds pairs of genes that are similar based on correlation coefficients and sequentially groups these pairs into clusters. Partition clustering further breaks these groupings into fixed sets to minimize within group dissimilarity.

Functional gene network analysis

Functional and molecular networks from gene expression data were generated using IPA (Ingenuity Systems, CA). A cutoff of 2.0 fold-change was used to assign genes to Ingenuity's Knowledge Base. Using IPA analysis, gene expression changes were grouped according to interacting gene networks that considered a context of physical, transcriptional, or enzymatic interaction between gene products.

Validation of selected genes from each cluster

The expression levels of selected genes were verified by real-time PCR analysis by Dr. Jin Nam as described previously.126 Briefly, Trizol-extracted RNA was converted to cDNA using the Superscript III Reverse Transcriptase Kit (Invitrogen, CA), and gene expression was determined by iCycler iQ real-Time PCR System (Bio-Rad, CA) using custom primers. Real-time PCR data was analyzed by ANOVA with Tukey's HSD post hoc test using SPSS v 17.

Results

Temporal regulation of gene expression during exercise

Principal components analysis (PCA) showed that global gene expression among the samples in the control and 15 day groups (n =3) was relatively uniformly distributed, while there was overlap in gene expression in the 2 or 5 day exercised cartilage samples

(Figure 3a). Hierarchical cluster analysis of the differentially regulated genes (p<0.05) indicated that distinct sets of genes were temporally regulated during each day of exercise

(Figure 3b). Of the 27,342 transcripts detected by the Affymetrix GeneChips array, 1720

(6.3%), 1748 (6.4%), and 2018 (7.4%) transcripts were significantly (p<0.05) and differentially up- or downregulated by more than 1.5-fold at 2, 5, and 15 days, respectively (Figure 3c). Compared to the control animals, the maximal change in gene expression occurred in the 15 day exercise animals. Gene expression was similar in the 2 day and 5 day animals. Distinct sets of genes were temporally regulated after exercise.

Interestingly, not all animals tested responded similarly.

Cluster analysis of genes temporally regulated during exercise

Partition clustering assigned regulated genes to one of nine clusters that showed trends in temporal gene regulation (Figure 4). The graphs represent the ten most regulated genes in each cluster; Cluster 5 contains only six genes. Genes in each cluster showed upregulation after days 2 and 5 of exercise followed by downregulation on day

15 (Cluster I), downregulation on day 2 followed by further downregulation on day 15

(Cluster II), upregulation on day 2 followed by a near leveling off (Cluster III), upregulation on day 2 followed by further upregulation on day 15 (Cluster IV), downregulation on day 2 or 5 followed by upregulation on day 15 (Cluster V), downregulation on day 2 followed by near leveling off or further downregulation (Cluster

VI), level or slight upregulation on days 2 and 5 followed by upregulation on day 15

(Cluster VII), downregulation on day 2 followed by upregulation on day 15 (Cluster

VIII), and downregulation on day 2 followed by leveling off or slight upregulation

(Cluster IX).

Figure 3. Gene Expression from Rat Cartilage Samples

Figure 3a. Principal components analysis of rat cartilage global gene expression. Samples in the control and 15 day exercised groups (n =3) were relatively uniformly distributed, whereas there was overlap in gene expression in the 2 or 5 day exercised cartilage samples. Figure 3b. Hierarchical cluster analysis showing the genes that were significantly (p<0.05) and differentially up- or downregulated at one or more time points by greater than two fold. The cluster map shows that distinct sets of genes were temporally regulated during each day of exercise. Gene expression was (Continued) 24

(Figure 3 continued) similar in 2 and 5 day exercised animals (n=3), and maximal change in gene expression occurred in the 15 day exercised animals compared to the controls.

Figure 3c. Graph showing the 926 transcripts two-fold or more differentially regulated of the 27,342 transcripts detected by the Affymetrix GeneChips array. In response to exercise, 339 genes were significantly upregulated, and 587 genes were downregulated.

Figure produced in collaboration with Dr. Jin Nam.

Figure 4. Clusters showing Trends in Temporal Gene Expression

Partition clustering by Partek assigned regulated genes to one of nine clusters that showed trends in temporal gene regulation. The graphs represent up to ten most regulated genes (two-fold or greater change) in each cluster. Graphs represent the relative changes in each cluster. Figure produced in collaboration with Dr. Jin Nam.

Expression of major molecular networks involved in cartilage in response to exercise

Among the nine gene clusters, IPA identified two gene networks associated with skeletal and muscular development and function (Clusters I and IV, Figure 5), two associated with cell cycle (Clusters II and VI, Figure 6), two associated with carbohydrate metabolism (Clusters III and VII, Figure 7), and one network associated with cellular development (Cluster III, Figure 8), immune cell trafficking (Cluster V,

Figure 9), connective tissue development (Cluster VI, Figure 10), skeletal and muscular disorder (Cluster VIII, Figure 11), cellular growth and proliferation (Cluster IX, Figure

12), and inflammatory response (Cluster IX, Figure 13).

In addition to the IPA analysis, tables containing the most significantly up- or down-regulated genes were constructed and analyzed manually to determine patterns in the data (Tables 1-8). IPA software typically uses networks based on cancer research; thus, a manual analysis of the data was used to avoid this potential analysis bias. Based on similar biological functions, exercise-regulated genes were manually categorized into five functional clusters: (Cluster I, Tables 1-3), Extracellular matrix (ECM) synthesis and degradation; (Cluster II, Table 4) Cell metabolism; (Cluster III, Tables 5-6) Cytoskeleton, ion channels and focal adhesions, (Cluster IV, Table 7) Cell signaling, growth, differentiation and proliferation, and (Cluster V, Table 8) Inflammation.

Manual cluster analysis of gene regulation

Cluster I. ECM synthesis and degradation

Notably, gene networks associated with the cartilage ECM were regulated by exercise, and the extent of up- or down-regulation of these genes is provided in Tables 1-

3. Genes encoding proteins involved in matrix synthesis or structure were upregulated, whereas genes that degrade the matrix were downregulated. For example, exercise significantly upregulated genes coding for proteins involved in cartilage scaffolding, elastin fiber synthesis, cell/matrix interactions, and proteoglycan synthesis, such as Cilp,

Eln, Vcan and Cytl1. Keratin sulfate genes, Chst1 and Chst3, and the heparin gene

Hst3st1, which code for structural components of cartilage, also were upregulated.

Suppressed genes included those coding for proteins involved in degradation of collagens, Mmp3, Mmp8, Mmp9, and Mmp14, as well as enzymes that degrade proteoglycans, such as Adamts3 and Adamts14. Conversely, genes encoding inhibitors of cartilage-degrading proteolytic enzymes, such as Serpina1, Serpina3n, Mug1, Mug2, Agt, and Timp4, were upregulated.

In support of cartilage phenotypic maintenance, transcription of genes that are not associated with cartilage tissue were suppressed. For example, the non-articular cartilage genes, Matn1, Matn3, Matn4, Col24a1, Col9a2, Col9a3, and Col1a2, were all downregulated by exercise. Similarly, a gene involved in bone remodeling and resorption, Ctsk, also was suppressed, and a negative regulator of bone formation, Pstn

(predicted), was also markedly downregulated.

Figure 5. Molecular Networks Generated by IPA from the Skeletal and Muscular

Development and Function Genes in Clusters I and IV

Red and white colors represent upregulation and no regulation as compared to CON cartilage, respectively. The shading of each color represents fold change in gene expression: dark = greater change and light = lesser change. Figure produced in collaboration with Dr. Jin Nam.

Figure 6. Molecular Networks Generated by IPA from the Cell Cycle Genes in

Clusters II and VI.

Green and white colors represent downregulation and no regulation as compared to CON cartilage, respectively. The shading of each color represents fold change in gene expression: dark = greater change and light = lesser change. Figure produced in collaboration with Dr. Jin Nam. 29

Figure 7. Molecular Networks Generated by IPA from the Carbohydrate

Metabolism Genes in Clusters III and VII.

Figure 8. Molecular Networks Generated by IPA from the Cellular Development

Genes in Cluster III.

Figure 9. Molecular Networks Generated by IPA from the Immune Cell Trafficking

Genes in Cluster V.

Figure 10. Molecular Networks Generated by IPA from the Connective Tissue

Development Genes in Cluster VI.

Figure 11. Molecular Networks Generated by IPA from the Skeletal and Muscular

Disorder Genes in Cluster VIII.

Figure 12. Molecular Networks Generated by IPA from the Cellular Growth and

Proliferation Genes in Cluster IX.

Green and white colors represent downregulation and no regulation as compared to CON cartilage, respectively. The shading of each color represents fold change in gene expression: dark = greater change and light = lower change. Figure produced in collaboration with Dr. Jin Nam. 35

Figure 13. Molecular Networks Generated by IPA from the Inflammatory Response

Genes in Cluster IX.

Green and white colors represent downregulation and no regulation as compared to CON cartilage, respectively. The shading of each color represents fold change in gene expression: dark = greater change and light = lower change. Figure produced in collaboration with Dr. Jin Nam.

Table 1. Extracellular Matrix Biosynthesis and Metabolism Genes Regulated in

Cluster I

Gene Day 0 Day 2 Day 5 Day 15 Gene Name Symbol Decorin Dcn 1 2.04 2.62 4.18 Fibroblast growth factor 2 Fgf2 1 2.65 2.84 2.73 Integrin 5 Itga5 1 1.76 1.97 2.09 Phospholipase C Plce1 1 4.18 4.02 4.21 Protein kinase C, alpha Prkca 1 1.75 1.97 1.94 Wnt 16 Wnt16 1 2.07 2.66 2.60 Syndecan Sdc4 1 2.05 1.87 2.23 Chondroitin sulfate N-acetylgalactosaminyltransferase 2 Csgalnact2 1 2.24 2.39 1.98 Chondroitin 6 Chst3 1 2.38 2.47 2.50 Mannosyl-glycoprotein b-1,4-N-acetylglucosaminyltransferase Mgat4a 1 1.93 2.71 2.08 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 St6gal1 1 2.85 2.81 2.29 Keratan sulfate Gal-6 sulfotransferase 1 Chst1 1 2.44 3.52 2.67 C1GALT1-specific chaperone 1 C1galt1c1 1 1.88 1.81 2.49 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 St6gal1 1 2.85 2.81 2.29 Heparan sulfate 3-O-sulfotransferase 1 Hs3st1 1 2.82 3.08 3.67 Versican Vcan 1 3.47 3.14 4.01 Carbohydrate (Keratan Sulfate Gal-6) Sulfotransferase 1 Chst1 1 2.44 3.52 2.67 Carbohydrate (Keratan Sulfate Gal-6) Sulfotransferase 3 Chst3 1 2.38 2.47 2.50 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 Hs3st1 1 2.82 3.08 3.67 Cytokine-like 1 Cytl1 1 4.52 5.06 5.71 Elastin Eln 1 1.86 1.41 1.52 Cartilage intermediate layer protein Cilp_ pred 1 1.99 2.08 2.47 Cartilage intermediate layer protein 2 Cilp2_ pred 1 1.76 1.90 2.35 CS N-acetylgalactosaminyltransferase-1 Csgalnact2 1 2.24 2.40 1.97 Matrilin 4 Matn4_ pred 1 -2.90 -3.11 -2.82 Podocan like 1 Podnl1 1 -3.71 -3.95 -4.12 Collagen, Type XXIV, Alpha 1 Col24a1 1 -2.12 -2.02 -2.32 Collagen, Type IX, Alpha 2 Col9a2_ pred 1 -2.16 -2.35 -2.23 Proteoglycan 2 Prg2 1 -4.56 -3.87 -4.30 Matrilin 3 Matn3_ pred 1 -5.95 -8.91 -12.28 Tenascin N Tnn _pred 1 -2.98 -2.19 -1.63 Collagen type 1a2 Col1a2 1 -2.03 -2.00 -1.41 Matrilin 1 Matn1 1 -1.85 -1.72 -2.33 Collagen, Type IX, Alpha 3 Col9a3 _pred 1 -1.95 -2.23 -2.18 Periostin, osteoblast specific factor Postn _pred 1 -4.11 -3.70 -1.97

Table 2. Inhibitors of ECM Degradation Genes Regulated in Cluster I

Gene Day 0 Day 2 Day 5 Day 15 Gene Name Symbol Serpin peptidase inhibitor, clade A (antitrypsin) Serpina1 1 4.86 12.15 12.36 Serpin Peptidase Inhibitor, Clade A memb3 Serpina3n 1 4.79 4.37 5.05 Angiotensinogen (Serpin Peptidase Inhibitor, Clade A 8) Agt 1 3.17 2.88 2.71 Murinoglobulin 1 Mug1 1 2.69 2.74 3.00 Murinoglobulin 2 Mug2 1 2.57 3.28 3.01 Mannan-Binding Lectin Serine Peptidase 1 Masp1 1 2.60 2.85 2.59 TIMP metallopeptidase inhibitor 4 Timp4 1 1.51 1.46 1.71 Serpin Peptidase Inhibitor, Clade A Serpinb1a 1 -4.62 -4.04 -2.40 Alpha-2-macroglobulin A2m 1 -2.17 -2.13 -2.47 Serpin Peptidase Inhibitor, Clade F Serpinf1 1 -3.55 -3.21 -1.34 Serpin Peptidase Inhibitor, Clade B Serpinb6b 1 -1.93 -2.04 -1.71

Table 3. ECM Remodeling Enzymes Regulated in Cluster I

Day 0 Day 2 Day 5 Day 15 Gene Name Gene Symbol Cathepsin K Ctsk 1 -2.86 -3.58 -2.48 ADAM metallopeptidase thrombospondin type1 motif, 3 Adamts3 1 -3.00 -4.36 -4.98 Matrix metallopeptidase 8 (neutrophil collagenase) Mmp8 1 -2.42 -3.17 -2.37 Carboxypeptidase Z Cpz 1 -4.04 -5.29 -5.35 Dipeptidyl-peptidase 4 Dpp4 1 -3.81 -3.87 -4.03 Matrix metallopeptidase 9 (gelatinase B) Mmp9 1 -8.20 -11.32 -7.84 ADAM metallopeptidase thrombospondin type1 motif, 14 Adamts14_ pred 1 -2.47 -2.36 -2.47 ADAM metallopeptidase thrombospondin type1 motif, 3 Adamts3 1 -2.48 -3.90 -3.31 Matrix Metallopeptidase 3 Mmp3 1 2.06 2.13 1.53 Matrix Metallopeptidase 14 Mmp14 1 -1.92 -2.50 -2.02 Heparanase Hpse 1 1.27 1.05 2.01

Table 4. Biosynthesis of Intermediate Metabolite Genes Regulated in Cluster II

ATPase, Na+/K+ transporting Atp1a2 1 2.16 1.61 1.59 Branched chain amino acid transaminase 1 Bcat1 1 1.65 1.96 2.19 Hexokinase 2 Hk2 1 2.16 1.36 2.61 NADH dehydrogenase (ubiquinone) 1 alpha Ndufa10 1 1.62 1.48 2.36 UDP-glucose pyrophosphorylase 2 Ugp2 1 1.81 1.62 2.02 B-cell CLL/lymphoma 2 Bcl2 1 1.96 2.25 2.70 DnaJ (Hsp40) homolog, subfamily A, member 1 Dnaja1 1 1.80 2.01 1.47 Heat shock protein 90, alpha, class A Hsp90aa1 1 2.09 2.10 2.11 Heat shock 70kD protein 1B Hspa1b 1 1.54 1.99 3.43 Heat shock 105/110 protein 1 Hsph1 1 3.38 3.18 3.00 Hypoxia up-regulated 1 Hyou1 1 1.87 1.82 2.07 Mitogen activated protein kinase 10 Mapk10 1 1.70 1.66 1.50 Glutamine-fructose-6-phosphate transaminase 2 Gfpt2 1 2.06 2.21 3.33 Hexokinase 2 Hk2 1 2.16 1.36 2.61 UDP-N-acteylglucosamine pyrophosphorylase 1 Uap1 1 1.73 1.94 2.75 UDP-glucose pyrophosphorylase 2 Ugp2 1 1.81 1.62 2.02 6-phosphofructo-2-kinase Pfkfb3 1 2.29 1.76 1.56 Argininosuccinate lyase Asl 1 1.63 2.09 2.06 Choline dehydrogenase Chdh 1 1.67 1.84 2.55 Amine oxidase, copper containing 3 Aoc3 1 2.30 2.21 2.89 Tyrosine hydroxylase Th 1 1.55 1.83 2.05 Glutathione S-transferase mu 3 Gstm3 1 1.92 2.13 2.87 Ceruloplasmin Cp 1 2.42 2.37 1.77 5-methyltetrahydrofolate-homocysteine methyltransferase Mtr 1 2.07 1.86 1.45 Phospholipase A2, group IIA Pla2g2a 1 1.03 1.87 9.74 Phosphatidic acid phosphatase type 2B Ppap2b 1 1.33 1.34 2.15 Phosphatidic acid phosphatase type 2B Ppap2c 1 1.28 1.49 2.06 Lipin 1 Lpin1 1 2.59 2.01 1.65 Glycosylphosphatidylinositol specific phospholipase D1 Gpld1 1 2.05 2.31 2.14 Phospholipase C, beta 1 Plcb1 1 2.00 1.98 1.92 Phospholipase C, epsilon 1 Plce1 1 4.18 4.02 4.21 ATPase, Na+/K+ transporting, alpha 2 polypeptide Atp1a2 1 2.16 1.61 1.59 Cubilin Cubn 1 2.53 2.66 2.29 5-methyltetrahydrofolate-homocysteine methyltransferase Mtr 1 2.07 1.86 1.45 Serine/threonine kinase 32B Stk32b_ pred 1 8.40 11.27 10.84 Serine/threonine kinase 32B Stk32b_ pred 1 7.34 9.57 8.43 Flavin-containing monooxygenase Fmo3 1 4.08 4.82 3.90 Alcohol Dehydrogenase, Iron Containing Adhfe1 1 2.66 2.72 2.67 Serotonin receptor 1B Htr1b 1 4.51 5.90 4.32 Protein kinase D1 Prkd1 1 2.42 2.42 2.02 Protein phosphatase 1, regulatory subunit 3C Ppp1r3c 1 2.82 2.80 2.55 Membrane Metallo-Endopeptidase Mme 1 -2.60 -3.04 -3.17 Carbonic anhydrase II Ca2 1 -4.15 -4.31 -5.69 Carbonic anhydrase I Car1_ pred 1 -7.11 -6.93 -6.42 Carbonic anhydrase III Car3 1 -3.37 -2.39 -1.75 Transferrin Trf 1 -3.65 -3.43 -2.24 Phospholipase A2, Group VII Pla2g7 1 -2.43 -3.27 -3.20 39

Table 5. Cell Signaling, Growth, and Differentiation Genes Regulated in Cluster III

Day 0 Day 2 Day 5 Day 15 Gene Name Gene Symbol Fibroblast growth factor 13 Fgf13 1 2.05 2.12 2.44 Fibroblast growth factor 14 Fgf14 1 2.19 2.35 2.66 Fibroblast growth factor 2 Fgf2 1 2.65 2.84 2.73 B-cell CLL/lymphoma 2 Bcl2 1 1.96 2.25 2.70 Guanine nucleotide binding protein Gng11 1 2.00 1.83 1.32 Heat shock protein 90, alpha Hsp90aa1 1 2.09 2.10 2.11 Protein phosphatase 2, regulatory subunit B Ppp2r3a 1 1.56 1.49 1.45 Protein kinase C, alpha Prkca 1 1.75 1.97 1.94 Thrombospondin 2 Thbs2 1 3.77 3.99 3.95 Calmodulin-like 3 Calml3 1 2.74 2.81 2.58 Phospholipase A2, group IIA Pla2g2a 1 1.03 1.87 9.74 Phospholipase A1 member A Pla1a 1 2.25 2.70 3.14 Phospholipase C Plce1 1 4.18 4.02 4.21 Heat shock protein B1 Hspb1 1 2.97 5.40 6.26 Heat shock 70kD protein 1B Hspa1b 1 1.54 1.99 3.43 Interleukin 1 receptor, type I Il1r1 1 2.10 2.04 1.77 Interleukin 1 receptor, type II Il1r2 1 1.72 1.65 2.01 Mitogen activated protein kinase 10 Mapk10 1 1.70 1.66 1.50 Protein kinase D1 Prkd1 1 2.42 2.42 2.02 Signal-induced proliferation-associated 1 like 2 Sipa1l2 1 1.92 2.55 2.16 ATPase, Na+/K+ transporting, alpha 2 polypeptide Atp1a2 1 2.16 1.61 1.59 FXYD domain-containing ion transport regulator 1 Fxyd1 1 1.57 1.73 2.41 5-hydroxytryptamine (serotonin) receptor 1B Htr1b 1 4.51 5.90 4.32 Phosphodiesterase 3A, cGMP inhibited Pde3a 1 2.04 2.14 2.16 Phosphodiesterase 3B, cGMP-inhibited Pde3b 1 2.62 2.28 1.74 Ciliary neurotrophic factor receptor Cntfr 1 2.59 3.09 3.55 Interleukin 23 receptor Il23r 1 3.62 2.28 1.63 ATPase, Na+/K+ transporting, alpha 2 polypeptide Atp1a2 1 2.16 1.61 1.59 Potassium inwardly-rectifying channel, subfamily J Kcnj8 1 1.60 1.83 2.02 Wingless-type MMTV integration site family, member 16 Wnt16 1 2.07 2.66 2.60 Protein phosphatase 2, regulatory subunit B'', alpha Ppp2r3a 1 1.56 1.49 1.45 Neurotrophic tyrosine kinase, receptor, type 3 Ntrk3 1 3.11 3.50 2.95 Inhibitor of DNA binding 2 Id2 1 2.45 2.02 1.55 Inhibitor of DNA binding 4 Id4 1 1.59 1.42 2.01 Cryptochrome circadian clock 1 Cry1 1 1.65 2.47 1.50 Cryptochrome circadian clock 2 Cry2 1 2.12 2.28 2.70 Period circadian clock 2 Per2 1 1.49 2.44 2.14 Interleukin 33 Il33 1 1.03 1.08 2.99 Bone morphogenetic protein 6 Bmp6 1 2.24 2.10 2.77 Transforming growth factor, beta 1 Tgfb1 1 2.00 2.05 2.48 Neurotrophic tyrosine kinase, receptor, type 3 Ntrk3 1 3.11 3.50 2.95 Heat shock 22kDa protein 8 Hspb8 1 3.47 3.51 3.73 Gremlin 1, DAN family BMP antagonist Grem1 1 3.53 2.59 2.06 Clusterin Clu 1 2.71 2.71 3.20 Leucine-rich repeat containing G protein-coupled recep 6 Lgr6 1 2.53 2.57 2.32 Zinc finger and BTB domain containing 16 Zbtb16 1 4.67 4.78 3.37 Chordin-like 2 Chrdl2_ pred 1 3.15 3.02 1.59 Phosphoinositide-3-kinase interacting protein 1 Pik3ip1 1 2.15 2.34 2.23 Phospholipase C1 Plce1 1 4.18 4.02 4.21 G protein-coupled receptor 75 Gpr75 1 2.32 2.36 2.08 RAS-Like, Family 12 Rasl12_ pred 1 3.08 2.52 3.52 Bone morphogenetic protein 5 Bmp5_ pred 1 1.69 1.54 1.53 Transforming growth factor, beta receptor III Tgfbr3 1 1.95 1.78 2.33 Connective tissue growth factor Ctgf 1 1.53 1.81 2.06 Follistatin-like 3 (secreted glycoprotein) Fstl3 1 1.63 1.47 1.57 FXYD domain-containing ion transport regulator 1 Fxyd1 1 1.57 1.73 2.41 Carboxypeptidase Z Cpz 1 -4.04 -5.29 -5.35 Bone morphogenetic protein 3 Precursor (BMP-3) Bmp3 1 -2.68 -2.77 -2.65 40

Table 6. Cell Cycle Genes Regulated in Cluster III

Day 0 Day 2 Day 5 Day 15 Gene Name Gene Symbol S100 calcium binding protein A9 S100a9 1 -7.37 -6.43 -3.65 Cyclin A2 Ccna2 1 -3.74 -3.73 -3.56 Cyclin B1 Ccnb1 1 -2.58 -2.68 -2.42 Cyclin B2 Ccnb2 1 -3.40 -3.44 -2.94 Cancer susceptibility candidate 5 Casc5 1 -2.97 -3.88 -3.15 Insulin-like growth factor binding protein 6 Igfbp6 1 -1.93 -1.38 3.96 Insulin-like growth factor 1 (somatomedin C) Igf1 1 -1.97 -2.42 -1.64 Bone morphogenetic protein 1 Bmp1 1 -1.90 -1.96 -1.86 Platelet-derived growth factor beta polypeptide Pdgfb 1 -2.12 -2.41 -1.73 Glycoprotein (transmembrane) nmb Gpnmb 1 -3.08 -3.82 -1.73 Caspase 1, apoptosis-related cysteine peptidase Casp1 1 -2.27 -2.15 -1.22 Tetraspanin 8 Tspan8 1 -2.27 -2.16 -1.72 Early growth response 1 Egr1 1 -2.89 -2.78 1.10 Heat shock protein 90kDa alpha (cytosolic), class A member 1 Hsp90aa1 1 2.09 2.10 2.11 Deoxynucleotidyltransferase Dntt 1 -2.96 -3.19 -3.53

Cluster II. Cell metabolism

Biomechanical signals generated by exercise regulated gene networks that control carbohydrate, protein, and fat metabolism, as well as ion channels and cytoskeletal proteins that would support intracellular transport and increase metabolism (Table 4).

For examples, exercise upregulated oxidative metabolism by upregulating Krebs cycle enzyme genes, such as Adhfe1, and Fmo3, a gene coding for an enzyme involved in

NADPH-dependent oxygenation of various nitrogen-, sulfur-, and phosphorous compounds. The phospholipase gene, Pld5, was among the most upregulated by exercise, as were multiple genes encoding molecules associated with membrane lipid and protein metabolism, including Itga5, Sdc4, Ppp1r3c, Plce1, Pla1a, Pde10a, and Pde3a.

Significantly downregulated enzyme-encoding genes included Trf, which is involved in

iron metabolism, and the MAP kinases Map4k1, and Pla2g7, which are involved in phospholipid hydrolysis.

Cluster III. Cytoskeleton, ion channels and focal adhesions.

Genes associated with the actin cytoskeleton as well as those controlling microtubule assembly were the major categories of cytoskeletal genes that were regulated by exercise (Tables 5-6). The microtubule assembly related genes included Eml5,

Map1b, Gda, Calm3, and Calml3, and the actin associated proteins were Gsn and Tmod2.

Furthermore, exercise regulated genes associated with focal adhesions, which are regulators of actin dynamics,147 including the genes Actn3, Gsn, Tmod, PIP3, PI3K, and

Map4k1. Similar to what was observed in the ECM cluster, cytoskeletal genes associated with tissues other than cartilage (e.g., muscle and bone) were significantly suppressed in cartilage. These genes include Actn3, Tnnt1, Tnni1, Tnnc2, Cdh15, Myl1, Tpm1, Vdr, and

Pvalb. Finally, exercise also upregulated several ion channel genes (Clcn4-2, Tmem63c,

Casr, Casrl1).

Table 7. Cytoskeleton, Ion Channel, and Adhesion Molecule Genes Regulated in

Cluster IV

Day 0 Day 2 Day 5 Day 15 Gene Name Gene Symbol Fibroblast growth factor 13 Fgf13 1 2.05 2.12 2.44 Fibroblast growth factor 14 Fgf14 1 2.19 2.35 2.66 Fibroblast growth factor 2 Fgf2 1 2.65 2.84 2.73 Gelsolin Gsn 1 2.35 2.23 3.20 Integrin, alpha 5 Itga5 1 1.76 1.97 2.09 Syndecan 4 Sdc4 1 2.05 1.87 2.23 Thrombospondin 2 Thbs2 1 3.77 3.99 3.95 Heat shock 70kD protein 1B Hspa1b 1 1.54 1.99 3.43 Ret proto-oncogene Ret 1 5.92 6.61 4.99 Calmodulin-like 3 Calml3 1 2.74 2.81 2.58 Phospholipase C, beta 1 Plcb1 1 2.00 1.98 1.92 Protein kinase C, alpha Prkca 1 1.75 1.97 1.94 ATPase, Na+/K+ transporting, alpha 2 polypeptide Atp1a2 1 2.16 1.61 1.59 Clusterin Clu 1 2.71 2.71 3.20 RT1 class II, locus Bb RT1-Bb 1 1.29 1.32 2.19 Purinergic receptor P2X, ligand-gated ion channel, 5 P2rx5 1 3.65 4.23 3.58 Syndecan Sdc4 1 2.05 1.87 2.23 Versican Vcan 1 3.47 3.14 4.01 Echinoderm Microtubule Associated Protein Like 5 Eml5 1 5.95 6.31 5.06 Plakophilin 1 Pkp1 1 3.22 3.99 5.13 Microtubule-Associated Protein 1B Map1b 1 3.63 4.27 3.68 Gelsolin Gsn 1 2.35 2.23 3.20 Guanine Deaminase Gda 1 2.89 3.61 3.97 Calsyntenin 2 Clstn2 1 3.38 3.19 3.50 Calmodulin 3 (phosphorylase kinase, delta) Calm3 1 2.74 2.81 2.57 Calmodulin-like 3 Calml3 1 2.74 2.81 2.58 Tropomodulin 2 Tmod2 1 1.86 2.13 3.27 Voltage sensitive chloride channel Clcn4-2 1 2.93 2.61 2.88 Transmembrane protein 63C Tmem63c 1 2.98 2.28 2.04 Calcium-sensing receptor Casr 1 2.16 2.05 2.62 FXYD domain-containing ion transport regulator 1 Fxyd1 1 1.57 1.73 2.41 Vomeronasal 2 receptor 1 pseudogene Casrl1 1 2.55 2.21 2.42 FXYD domain-containing ion transport regulator 1 Fxyd1 1 1.57 1.73 2.41 N-Cadherin Cdh2 1 -3.27 -3.47 -3.25 Troponin T type 1 Tnnt1 1 -2.78 -2.62 -2.90 Stathmin1 Stmn1 1 -4.27 -4.39 -3.77 Phosphatidylinositol-4-phosphate 5-kinase, type I, beta Pip5k1b 1 -2.70 -3.19 -2.95 Vitamin D (1,25- dihydroxyvitamin D3) receptor Vdr 1 -3.33 -3.88 -3.36 Actinin, Alpha 3 Actn3 1 -2.13 -2.81 1.12 Troponin I Type 1 Tnni1 1 -1.85 -1.81 -2.10 Troponin C2 Tnnc2 1 -2.74 -3.08 1.28 Actin, Alpha 1 Acta1 1 -5.10 -5.71 -1.62 M-Cadherin Cdh15 1 -2.26 -1.96 -2.43 Myosin, Light Chain 1 Myl1 1 -4.45 -6.24 -1.23 Tropomyosin 1 Tpm1 1 -2.25 -2.81 -1.94 Parvalbumin Pvalb 1 -2.04 -2.88 1.58 43

Table 8. Inflammation Related Genes Regulated in Cluster V

Day 0 Day 2 Day 5 Day 15 Gene Name Gene Symbol Chemokine (C-X-C motif) ligand 13 Cxcl13 1 2.25 4.37 9.85 Interleukin 16 Il16 1 2.62 3.07 2.69 Interleukin 1 receptor, type II Il1r2 1 1.72 1.65 2.01 Interleukin 17B Il17b 1 1.83 2.24 2.02 Interleukin 1 receptor, type I Il1r1 1 2.10 2.04 1.77 Interleukin 6 receptor Il6ra 1 2.18 2.61 1.92 Lipopolysaccharide binding protein Lbp 1 1.80 1.89 3.00 Angiotensinogen (Serpin Peptidase Inhibitor, Clade 8) Agt 1 3.17 2.88 2.71 Murinoglobulin 1 Mug1 1 2.69 2.74 3.00 Murinoglobulin 2 Mug2 1 2.57 3.28 3.01 Mannan-Binding Lectin Serine Peptidase 1 Masp1 1 2.60 2.85 2.59 Carboxypeptidase, vitellogenic-like Cpvl 1 2.96 2.72 2.91 ST6 Beta-Galactosamide Alpha-2,6-Sialyltranferase 1 St6gal1 1 2.85 2.81 2.29 Thrombospondin 2 Thbs2 1 3.77 3.99 3.95 Plasminogen activator, tissue Plat 1 2.06 2.37 4.25 FK506 binding protein 5 Fkbp5 1 6.50 6.70 8.11 Clusterin Clu 1 2.71 2.71 3.20 Phosphodiesterase 10A Pde10a 1 2.58 2.34 2.61 Phosphodiesterase 3A Pde3a 1 2.04 2.14 2.16 Phospholipase D 5 Pld5 1 11.19 12.71 8.19 Phospholipase C1 Plce1 1 4.18 4.02 4.21 Phospholipase a1 Pla1a 1 2.25 2.70 3.14 Phosphodiesterase 3B Pde3b 1 2.62 2.28 1.74 Guanine nucleotide binding protein (G protein), gamma 11 Gng11 1 2.00 1.83 1.32 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 Pfkfb3 1 2.29 1.76 1.56 Phospholipase A2, group IIA Pla2g2a 1 1.03 1.87 9.74 Cathepsin S Ctss 1 1.05 1.27 2.86 Heat shock 22kDa protein 8 Hspb8 1 3.47 3.51 3.73 Heat shock protein 90kDa alpha (cytosolic), class A 1 Hsp90aa1 1 2.09 2.10 2.11 Heat Shock protein 105 Hsph1 1 3.38 3.17 3.00 Heat shock 70kDa protein 1B Hspa1b 1 1.54 1.99 3.43 Angiopoietin-like 1 Angptl1 1 2.18 1.79 2.16 Gelsolin Gsn 1 2.35 2.23 3.20 Cathepsin S Ctss 1 1.05 1.27 2.86 Jun B proto-oncogene Junb 1 -1.62 -1.50 2.12 Tumor necrosis factor receptor superfamily, member 11b Tnfrsf11b 1 1.84 1.83 2.27 Plasminogen activator, tissue Plat 1 2.06 2.37 4.25 Serpin peptidase inhibitor, clade A Serpina1 1 4.86 12.15 12.36 Serpin peptidase inhibitor, clade A member 3N Serpina3n 1 4.79 4.37 5.05 Complement component 1, q subcomponent, C chain C1qc 1 -1.34 -1.10 2.14 Chemokine (C-X-C motif) receptor 4 Cxcr4 1 -3.30 -3.10 -2.65 Arachidonate 15-lipoxygenase Alox15 1 -2.32 -2.06 -2.81 Prostaglandin-endoperoxide synthase 2 Ptgs2 1 -2.24 -2.57 -2.15 Complement component 3 C3 1 -3.50 -2.81 -2.05 Complement component 7 C7 1 -2.66 -3.69 -2.12 Serglycin Srgn 1 -4.08 -4.26 -3.27 Neutrophilic granule protein Ngp_ pred 1 -7.07 -6.77 -3.86 Kelch-Like Family Member 6 Klhl6_ pred 1 -4.86 -5.36 -3.43 Proteoglycan 2 Prg2 1 -4.56 -3.87 -4.30 Interferon regulatory factor 8 Irf8 1 -2.31 -2.78 -1.72 Defensin, alpha 1 Defa 1 -7.36 -6.30 -1.81 Colony stimulating factor 1 receptor Csf1r 1 -3.24 -3.16 -1.56 CD93 molecule C1qr1 1 -2.24 -2.65 -1.35 Lysozyme Lyz 1 -4.23 -4.31 1.19 Chemokine (C-X-C motif) receptor 2 Il8rb 1 -2.07 -1.76 -1.82 Chemokine (C-C motif) receptor 1 Ccr1 1 -2.29 -3.04 -1.79 Chemokine (C-X-C motif) ligand 12 Cxcl12 1 -2.35 -2.97 -1.82 Dipeptidyl-peptidase 4 Dpp4 1 -3.81 -3.87 -4.03 Serpin Peptidase Inhibitor, Clade A Serpinb1a 1 -4.62 -4.04 -2.40 Mitogen-activated protein kinase kinase kinase kinase 1 Map4k1_ pred 1 -2.23 -2.46 -2.30 TYRO protein tyrosine kinase binding protein Tyrobp 1 -3.22 -2.97 -2.65 Phosphoinositide-3-kinase adaptor protein 1 Pik3ap1_ pred 1 -3.47 -3.79 -3.14 Protein kinase, cGMP-dependent, type II Prkg2 1 -4.56 -4.22 -6.73 Coagulation factor V (proaccelerin, labile factor) F5 1 -1.86 -2.53 -1.65 High mobility group box 2 Hmgb2 1 -3.83 -3.59 -3.40 S100 calcium binding protein A8 S100a8 1 -4.54 -4.32 -1.98 Phospholipase A2, Group VII Pla2g7 1 -2.43 -3.27 -3.20 Phospholipase A2 Group Iva Pla2g4a 1 -1.88 -2.06 -1.83 Heat shock 22kDa protein 8 Hspb8 1 3.47 3.51 3.73 Heat shock protein 90kDa alpha (cytosolic), class A 1 Hsp90aa1 1 2.09 2.10 2.11 Heat Shock protein 105 Hsph1 1 3.38 3.17 3.00 Heat shock 70kDa protein 1B Hspa1b 1 1.54 1.99 3.43 Angiopoietin-like 1 Angptl1 1 2.18 1.79 2.16 Fos-B gene Fosb 1 -1.38 -1.54 6.08 44

Cluster IV. Cell signaling, growth, differentiation and proliferation

Genes controlling signaling pathways were the most highly regulated in response to exercise of all of the pathways studied (Table 7). These pathways and pathway components included PI3K-AKT, Ras, mitogen activated protein kinases (MAPK), Rap1,

Cyclic AMP, Dopaminergic synapses, Wnt, AMPK, Jak-STAT, ErbB, Inositol phosphate, VEGF, p53, FoxO, and mTOR. In addition to cell signaling, cell growth, differentiation, and proliferation genes were significantly regulated by exercise. For example, transforming growth factor (TGF) genes, including, Tgfb1, Bmp6, and Bmp5

(predicted), were upregulated by exercise, and the fibroblast growth factor genes upregulated included Fgf2, Fgf13, and Fgf14. It should be noted that these growth factors are regulated by the signaling pathways that were also regulated in response to exercise (e.g., Ras, Rap1, MAPK, Wnt, etc.). Other genes, including Prkd1, Ret, Ntrk3,

Hspb8, Cntfr, Gpr75, and Gprc5a, also contribute to signaling pathways involved in cell growth and differentiation. A gene involved in chondrocyte differentiation, Ctgf, also was upregulated.

Interestingly, exercise upregulated genes controlling cell growth and differentiation, but suppressed genes that enabled progression of the cell cycle. For example, the mitosis related genes S100a9, Ccna1, Ccna3, Ccnb1, Ccnb2, Casc5,

Hsp90aa1, Calm3, and Calml3, and cell proliferation/mitosis genes Pdgfb, Gpnmb,

Casp1, Tspan8, and Egr1, were all downregulated by exercise.

Cluster V. Inflammation

Exercise has been shown to both suppress and increase inflammation,148 and genes regulated by exercise in this study reflect that contradiction (Table 8). For example, exercise significantly upregulated genes encoding decoy receptors for IL-6

(Il6ra) and IL-1 (Il1r2); however, exercise significantly upregulated Il1r1, Il17b, and

Il16. Exercise also upregulated immune response-associated protease inhibitor genes such as Agt, Mug1, Mug2, Cpvl, clotting factor genes Thbs2 and Plat, and signaling molecule genes St6gal1, Fkbp5, Pde10a, Pde3a, Pde3b, Pld5, Plce1, and Pla1a, as well as a chemokine for B lymphocytes (Cxcl13), and Lbp, a gene involved the acute phase immune response. Also upregulated were multiple genes encoding heat shock proteins, including Hspb8, Hsp90aa1, Hsph1, Hspa1b. These genes are associated with stress responses, signaling control, and protein folding.

Conversely, exercise suppressed multiple inflammatory genes including those encoding chemokine ligands and receptors, such as Cxcl12, Cxcr1, Cxcr2, and Cxcr4, and those encoding genes for inflammatory signaling cascades, including Tyrobp,

Map4k1, and Prkg2. Exercise also inhibited arachidonate metabolism by suppressing expression of Alox15 and Ptgs2, as well as phospholipases (Pla2g7 and Pla24ga), and the complement cascade (C3 and C7). Exercise suppressed genes involved in both acute inflammation, such as Hmgb2 and S100a, but suppressed a negative regulator of the innate immune system, Pik3ap1, which suppresses toll-like receptor (TLR) responses.

Finally, exercise suppressed genes responsible for enzymes found in inflammatory cells

that mediate the immune response. These genes include Srgn (mast cells), Ngp

(predicted, neutrophils), Defa (neutrophils), Dpp4 (T cells), and Csf1r (granulocytes).

Exercise regulates genes linked to OA pathology

As described above, exercise suppressed multiple genes that could be important in

OA pathology, including those involved in inflammation, matrix destruction, and cell cycle progression. Some genes identified as candidate OA genes by Human Genome

Epidemiology Navigator138 were regulated by exercise (Table 6). For example, exercise suppressed many genes that may be upregulated in OA, such as Col1a2, Matn3, Mmp8,

Mmp9, Ptgs2, Adamts3. Conversely, some potentially OA-associated genes, which may also be involved in normal ECM homeostatic processes such as remodeling, were upregulated by exercise (Cilp, Mmp3, Tgfb1, Timp4, Tnfrsf11b).

Discussion

To our knowledge, this is the first study to examine gene expression changes underlying cartilage homeostasis in response to exercise in normal, healthy tissue.

Microarray analysis was used to determine changes in gene expression in response to the beginning of a low intensity walking exercise program. One of the major findings of this study is that mechanical signals generated by exercise elicited a regenerative response in the cartilage that increases ECM synthesis and cellular metabolism to promote health and homeostasis of the tissue. At the same time, exercise suppressed chondrocyte division and proliferation, which are hallmarks of OA onset.149 Additionally, exercise regulates immune response genes to upregulate many anti-inflammatory genes and suppress many pro-inflammatory genes, and several genes expressed in OA are downregulated by

exercise. These results may provide a mechanism for the prevention of onset and delayed progression of OA by exercise, which is a known therapeutic treatment option for this disease.

For example, the IPA network analysis indicated that one of the major effects of exercise on cartilage is the maintenance of the cartilage ECM and the cartilage phenotype. This is unsurprising given that in vitro mechanical loading studies indicate that dynamic compression increases ECM synthesis.149 Integrins and other receptors on the chondrocyte surface respond to mechanical stimulation and also to fibronectin and

Type II collagen fragments in the ECM.149 While a healthy mechanical stimulus level leads to the production of proteinases, cytokines, and chemokines, injurious static compression leads to proteoglycan loss, damaged collagen, and reduces synthesis of

ECM proteins.149

In this study, low impact walking exercise induced the expression of cartilage associated ECM genes such as Vcan, Cilp, and Eln, but it markedly inhibited non- articular cartilage associated ECM genes such as Matn1, Col1a2, Prg2, Podnl1, and

Col9a2. Additionally, some of these non-articular cartilage associated genes downregulated by EX are found to be upregulated in osteoarthritic cartilage.150, 151, 152

Furthermore, many of the genes encoding the collagenases and aggrecanases that are detrimental to cartilage integrity, such as Mmp8, Mmp9, Mmp14, Adamts3, and

Adamts14, are significantly inhibited by exercise. Matrix metalloproteinases (MMPs) and aggrecanases degrade native collagens and proteoglycans and include the collagenases (MMP-1, MMP-8, and MMP-13), the gelatinases (MMP-2 and MMP-9),

stromelysin-1 (MMP-3), and membrane type I (MT1) MMP (MMP-14).149 MMP-14 produced by OA chondrocytes activates pro-MMP-13, which in turn cleaves pro-MMP-9.

Importantly, increased global gene expression of aggregan and type II collagen has been observed in human OA compared to healthy cartilage,149 so the fact that some genes encoding proteolytic enzymes are upregulated in this study (Cilp, Mmp3, Tgfb1, Timp4,

Tnfrsf11b) may indicate a healthy remodeling state of the cartilage.

At the same time, genes that code for inhibitors of matrix metalloproteinases, such as Serpina1, Serpina3, Mug1, Mug2, and Timp4, are upregulated to minimize cartilage proteolysis. Collectively, these gene expression alterations in response to exercise may be important for maintaining cartilage health.

Network analysis also showed that exercise upregulated cell metabolism and cytoskeletal components to support cellular biomechanics and nutrient uptake. This cluster contained cell membrane associated genes that are involved in cell surface mediated signaling, adhesion, and cell growth/differentiation, such as Itga5 and Sdc4, as well as a gene for phospholipases (Pla1a) that signal through the membrane and break down phospholipids. Exercise also upregulated phosphodiesterases (Pde3a, Pde3b,

Pde10a) and protein kinases (Stk32b, Prkd1) that are involved in signal transduction.

Exercise also induced some of the genes involved in the regulation of calcium sensing, flavin metabolism, and osmosensitive channels (Casr, Casrl1, Fmo3, Tmem63c), while suppressing genes for the carbonic anhydrases that are involved in acid-base balance

(Ca2, Car1, Car3). It is not clear how suppression of these enzymes benefits cartilage metabolism.

Another major effect of the exercise training was the upregulation of genes related to the cytoskeleton, including those encoding microtubules (Eml5, Map1b, Gda, Calm3), intermediate filaments (Pkp1), and calmodulin (Calm3), which is involved in microtubule assembly. Conversely, exercise suppressed a gene coding for a protein that inhibits microtubule assembly (Stmn1). It is not surprising that exercise would affect the cytoskeleton, as cytoskeletal rearrangements and synthesis are a known consequence of biomechanical signaling.153, 154 As a likely effort to maintain cartilage integrity and prevent intrusion of other tissues into the cartilage, exercise downegulated many genes found in other tissues, such as muscle. These genes included Tnnt1 and Tnnc2, which regulate actin-myosin complexes; Actn3, which is responsible for thin filament crosslinking; Cdh15, which is responsible for myotube formation; Tpm1, which stabilizes actin-tropomyosin; and Pvalb, which is involved in muscle relaxation.

Regeneration and maintenance of the ECM requires growth and differentiation factor expression. Thus, it was not surprising that exercise upregulated genes from the

TGF-β family of growth factors, including Bmp6, Tgfb1, and Bmp5. Conversely, exercise suppressed expression of Bmp1, a regulator of bone repair.155 Additionally, exercise induced the expression of fibroblast growth factors Fgf2, Fgf13, and Fgf14, and the gene for connective tissue growth factors, Ctgf, to promote cartilage regeneration.156,

157 Exercise also induced expression of Grem1 and Chrdl2, genes that may be required for tissue patterning and differentiation. These observations further underscore the importance of exercise in supporting cartilage tissue homeostasis and integrity.

Although exercise clearly exerts regenerative influences on cartilage, it inhibited several genes integrally associated with cell cycle progression. Among the most suppressed genes were S100a8 and S100a9. This suppression is important because these proteins have been shown to upregulate and activate MMPs and aggrecanases in mouse arthritis models.149 In fact, elevated levels of these two proteins may predict joint destruction in OA, and S100A9 in particular plays a major role in the mineralization of the cartilage matrix in hypertrophic chondrocytes.128

Genes coding for cyclins that are involved in activation of Cdk kinases, which are regulators of cell cycle and mitosis (Ccna1, Ccnb1, Ccnb2) were also highly suppressed.

Genes important in the regulation of cell division, such as Igf1, Igfbp6, Pdgfb, and Bmp1, were suppressed, along with a signal for chondrocyte proliferation, Serpinf1.158 Thus, exercise appeared to promote cell synthesis, but not proliferation. This finding is important because increased cell proliferation and ECM loss is observed in pathological cartilage, such as in OA.159

Exercise prevents the progression of OA in arthritic cartilage.127 In healthy cartilage, exercise upregulated genes for the decoy receptors for IL-6 and IL-1, but did not upregulate the genes for these cytokines. This pattern of gene expression indicates a dampening of inflammation. Similarly, exercise has been shown to upregulate toll-like receptor (TLR)-4 in circulating lymphocytes.160 However, a downregulation of genes that are ligands for toll-like receptors (Hmgb2 and S100a8) were observed in this study.

In total, exercise suppressed a significant number of genes that are upregulated during acute and chronic inflammation, including genes encoding chemokine ligands and

receptors (Cxcl12, Cxcr1, Cxcr2, and Cxcr4), arachidonate metabolites (Alox15 and

Ptgs2), phospholipase mediators of membrane signaling (Pla2g7 and Pla24ga), and the complement components (C3 and C7).126 Exercise also suppressed immune cell- associated genes, such as Srgn (mast cells), Ngp (predicted, neutrophils), Defa

(neutrophils), Dpp4 (T cells), and Csf1r (granulocytes). However, the function of these genes in cartilage is unclear. In summary, the alterations of genes known to control inflammatory cascades and influence the immune system by exercise suggests that exercise is a protective influence on the cartilage that protects it from inflammation and likely from the onset of OA. Collectively, inhibition of these molecules may be important in preventing initiation of clotting cascades and pro-inflammatory signaling activities. Nevertheless, the role of exercise on inflammation is a complicated one, and exercise did induce expression of some genes required for inflammatory cascades, including protease inhibitors (Agt, Mug1, Mug2, Cpvl) and clotting factors (Thbs2, Plat).

The increase in heat shock protein expression that we observed has been shown previously to be chondroprotective, as it may reduce stress-related apoptosis in chondrocytes.161

Exercise may play a role in preventing the onset and progression of OA.129, 162

Meta-analysis studies have identified several genes that are associated with cartilage afflicted with OA. Additionally, several biomarkers for OA have been predicted.163, 164,

138 Several genes associated with OA were present in all of the clusters, suggesting that

OA alters cartilage metabolism in addition to being an inflammatory disease. Exercise downregulated many OA associated genes,138 thus providing a mechanism for the

beneficial effects of exercise in prevention of onset and progression of OA. OA onset is characterized by an inability of the chondrocytes to maintain the balance between synthesis and degradation of the ECM. In this study, growth factors and enzyme inhibitors that promote ECM synthesis were upregulated. Catabolic factors, including some proinflammatory mediators and degradative enzymes, were suppressed. In a comparative proteomic analysis of articular cartilage from normal human donors and OA patients, MMP-2 was increased in OA cartilage, and cathepsin K upregulation coincided with cartilage damage.165 Similarly, here we observed downregulation of serglycin, a protein that mediates processing of MMP-2, as well as cathepsin K. In a recent genome- wide expression profiling study comparing normal and OA cartilage, expression of

Zbtb16, Igf1, Mmp9, and Col1a2 were differentially expressed.125 We observed the opposite expression pattern for these genes in our analysis of healthy cartilage. Finally, increases in genes involved in the endochondral ossification process characterize osteophytic chondrocytes.166 We observed an expression pattern opposite to the endochondral ossification process in Mmp9, Ca2, Col1a, and Grem1 genes.

Low impact walking in rats was used in this study to determine gene expression consequences of exercise training. The effects of high impact, high intensity exercise on gene expression should also be studied. Further, the exercise in this study was 45 min/day for 15 days; the consequences of longer exercise bouts or differing rest intervals between the exercise bouts should be determined. Finally, the effects of exercise on gene expression in other species should be examined.

Conclusions

While it has long been suspected that exercise is beneficial for cartilage health, the results of this study indicate that exercise effectively maintains cartilage matrix homeostasis by increasing cell metabolism, matrix synthesis, protease inhibitor expression, and cell growth, while decreasing potentially detrimental proteolytic and inflammatory processes. Exercise suppresses cell proliferation signals that lead to the onset of OA. Furthermore, it serves as an anti-inflammatory regulator by suppressing genes associated with acute inflammation and the complement and clotting systems, by suppressing cytokine and chemokine expression, as well as by suppressing signaling pathways involved in membrane activation and generation of arachidonate metabolites during inflammation. Exercise also directly suppresses several genes known to be upregulated in OA while upregulating genes suppressed in OA. These results provide a potential mechanism for the protective effects of exercise as a master regulator of gene activity to prevent the onset of OA in healthy articular cartilage.

Chapter 3: Exercise as Integrator of Bone and Muscle Health

Introduction

Mechanisms coordinating bone and muscle health are not well defined, but mounting evidence indicates that muscle and bone strengthening must be regulated in part by common mechanisms, and particularly in response to exercise.167, 168, 169, 170, 171

In addition to their physical interaction, bone and muscle may be in communication via circulating mediators. Such circulating factors produced by bone or muscle that can act on distant tissues have been recognized recently, and the response of bone to differing strains as a result of normal daily activity is evidence of this bone and muscle crosstalk.167 Providing support for the importance of circulating muscle-derived factors on skeletal health in the absence of mechanical stimulation are studies showing that muscle flaps can enhance bone formation, and muscle implants along periosteum can directly induce bone growth.168 Exercise further induces the release of osteogenic factors, insulin-like growth factor (IGF)-1 and fibroblast growth factor (FGF)-2, myostatin, and extracellular matrix factors from the muscle,168 and these and other factors may serve to sense, transduce, and translate biomechanical signals into relevant biochemical signals that can alter musculoskeletal health.169 In particular, inhibition of myostatin or growth and differentiation factor-8 (GDF-8, MSTN), an inhibitor of muscle hypertrophy, increases both bone mass in trabecular bone of the proximal humerus and cortical bone in the deltoid crest of mice, and increases the regenerative potential of both 55

bone and muscle in response to injury.172, 173, 174, 171 Inhibition of MSTN through various means, and in particular, by endogenous factors that are upregulated by exercise, could be a promising method for increasing bone and muscle growth. Therefore, the focus of this work is a molecular mechanism involving the MSTN protein and its interactions with other soluble mediators in response to exercise.

One such mediator is follistatin-like 3 (FSTL3), a novel circulating, mechanosensitive protein, which mediates exercise-driven bone formation.30 In addition,

FSTL3 binds to MSTN and prevents MSTN from binding to its receptor and therefore prevents the downstream cascades that increase muscle atrophy and decrease muscle hypertrophy. Thus, FSTL3 may also impact muscle formation and strengthening.

Because FSTL3 is upregulated by exercise, the interaction between FSTL3 and MSTN may provide insights into how exercise modifies musculoskeletal health. The following studies demonstrate that FSTL3 is a mediator of exercise-driven bone formation and strengthening, and that FSTL3 may also serve as a mediator linking bone and muscle.

FSTL3 as Mediator of Bone and Muscle Crosstalk

Like muscle, bone also requires mechanical signals for strengthening and adaptation to loading. Mechanical forces generated by exercise are converted into biochemical signals that are essential for bone adaptation. Networks of cells within the bone that translate these signals and regulate bone remodeling include osteoblasts (OB), osteocytes (OCY), and osteoclasts (OCL). All of these cells are mechanosensitive.175

Immunofluorescence analysis of rat femur cross sections following 2 or 5 days of exercise showed exercise-dependent induction of FSTL3 that was mostly localized to

OCY and trabecular bone lining OBs.30 FSTL3 has also been shown to inhibit differentiation of bone resorptive OCLs in vitro.176

FSTL3 mRNA expression in human muscle is significantly upregulated in response to a single bout of eccentric resistance exercise,177, 86 and in the trabecular and cortical bone as well as bone marrow of rat femurs.30 Gene-silencing studies in mice demonstrated definitively that FSTL3 is required for exercise-dependent bone formation.30 Bone mineral apposition rate (MAR) was estimated by sequential intraperitoneal (i.p.) administration of calcein and alizarin complexone on day 3 and day

12 after the start of exercise, respectively. The relatively narrow distance between the calcein and alizarin incorporation in the non-exercised wild-type (WT) and global FSTL3 gene deletion (KO) mice revealed limited bone deposition, whereas WT mice subjected to exercise demonstrated a significant increase in bone deposition on the endosteal surface of the posterior side of the femur. Such exercise-driven bone formation was not observed in FSTL3 KO mice. FSTL3 KO mouse bones also showed significantly lower stress at ultimate strength and lower fracture strain as compared to WT mice, indicating that FSTL KO bones are weaker than those from WT mice.

In response to 2 days of walking exercise, FSTL3 is significantly upregulated in the bone, cartilage, and (Vastus lateralis) muscle (Figure 1).30 As described previously, although published data point to FSTL3 as a collective regulator of bone and muscle growth and strength in response to exercise,30 it is possible that FST plays a role in this regulation as well. FST stimulates myogenic transcription factors in myoblasts, blocks

MSTN, activin, and TGF-β1,71 and potentially could compensate for deleted FSTL3 in binding MSTN at the tissue level.

Follistatin as Mediator of Bone and Muscle Crosstalk

Since cell-surface binding for the follistatin proteins is FST288 > FST303 >

FST315 > FSTL3, neutralization of MSTN has been shown to correlate with the relative abilities of the isoforms to bind cell-surface proteoglycans.33 Thus, FSTL3 is weaker at neutralizing MSTN than the FST isoforms at the cellular level, but in all cases the preferred ligand might be the one that is encountered first.33 Overexpression of FST in mice has been shown to produce even greater effects on muscle mass than MSTN null mice, suggesting that FST may be inhibiting a ligand other than MSTN; however, the identity of this ligand remains speculative (e.g., GDF-11, activins), and the effect may be the result of a process other than MSTN inhibition.65 However, since Fst gene deletion has more global effects on the body compared to Fstl3 deletion,47 we have chosen in this study to focus on FSTL3 for further research and development as a MSTN inhibitor. FST can modulate effects of activin, inhibin, follicle-stimulating hormone, and some BMPs, interrupting pituitary and gonadal function.178 Nevertheless, we have included the study of FST when possible to further clarify the roles of FSTL3 on the musculoskeletal system. One of the limitations of this work is that study of non-follistatin family molecules has not been included.

Exercise-Mediated Regulation of FSTL3 and MSTN

While the independent functions of MSTN and FSTL3 are known, few published studies have examined their combined regulation and effects on musculoskeletal health in

response to exercise, to our knowledge. Acute resistance exercise was shown to significantly increase FSTL3 and significantly downregulate MSTN mRNA in muscle biopsies,86 regardless of feeding status. Similarly, an acute bout of eccentric exercise significantly decreased MSTN and ActRIIB but significantly increased FST, FSTL3, and

GASP-1 in muscle biopsies.177 However, another study comparing the effects of resistance exercise on younger and older men revealed no change in FSTL3 mRNA after

12 days of training,179 and yet another study found no change in FSTL3 mRNA after a 21 week resistance training protocol.180 The results of these studies indicate that the changes in MSTN and FSTL3 are likely transient. Finally, after 12 weeks of lower body resistance training, both serum MSTN and FSTL3 were upregulated,79 indicating potential for differential responses in tissue versus serum. It should be noted that in none of these studies was aerobic or low intensity walking exercise tested, indicating that much is still unknown about the regulation of MSTN/FSTL3 in response to exercise.

The goal of this research was to determine a molecular mechanism that collectively regulates bone and muscle formation and strengthening in response to low- intensity walking exercise, specifically by determining the effects of the FSTL3/MSTN system on the musculature. This research will enhance fundamental understanding of the role of exercise in molecular regulation of muscle and bone health by investigating a key pathway known to affect the musculoskeletal system in response to exercise. Further, neutralization of MSTN by FSTL3 and FST shows promise for clinical applications in muscle disorders to increase muscle growth and strength.181, 182

Materials and Methods

Mouse models

All experiments conformed to the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health and were approved by the

Institutional Animal Care and Use Committee of The Ohio State University. Male wild- type mice (The Jackson Laboratory), age 12 weeks, were housed at constant temperature

(22 ± 2˚C) and provided with a 12-h light/dark cycle. Mice were given standard lab chow and water ad libitum. FST288 mice (n=5 mice per group) were developed and kindly provided by Dr. Alan Schneyer, and creation and characterization of these mice has been previously described.48 These mice resulted from a knock-in mutation was made on a mixed 129S4/SvJae × C57BL/6 background. Other transgenic mouse models compared to the C57BL/6 WT (n=10 mice per group) included a previously described

Fstl3-/- mouse model (“FSTL3 KO,” C57BL/6 background, n=5 mice per group)31 and

FSTL3/FST288 double knockout (DKO) mice (n=5/group).

Ex vivo assessment of skeletal muscle contractility

Muscles and contractility apparatus were prepared using methods previously described.183 Briefly, intact extensor digitorum longus (EDL) and soleus (SOL) muscles were surgically dissected and mounted vertically between two stimulating platinum electrodes and immersed in 50 mL bathing chambers containing modified Ringer solution consisting of 139 mM NaCl, 3 mM KCl, 17 mM NaHCO3, 12 mM glucose, 1 mM

MgCl2, and 2.5 mM Ca. Muscle chambers were supplied with pure oxygen during the entire protocol. A constant stimulatory voltage was applied to equilibrate muscles at

maximum force (80 hz pulses, repeated every minute for 30 minutes), followed by stimulation with a Grass S88X stimulator at frequencies from 1-150 hz to generate force vs. frequency curves and fatigue protocol as described previously.183 The force versus frequency curve was generated by stimulating EDL muscles at the following frequencies:

1, 5, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, and 150 hz. SOL muscles were stimulated up to 80 hz. For the fatigue protocol, a stimulus of 40 hz was repeated every 2 sec for 5 min. Contractile forces were detected using a force transducer integrated with an ADI

PowerLab 8/35 system. The soleus and EDL muscles were chosen for these experiments because they represent a slow-oxidative (type I and IIa muscle fibers) and fast-glycolytic muscle (type IIb and IIx muscle fibers), respectively.

Transient transfection of mouse myoblast cells with Fstl3

C2C12 murine myoblasts were obtained from the American Type Culture

Collection (ATCC Number: CRL-1772).184, 185 Approximately 1.5 x 106 C2C12 cells were seeded in each well of a 6-well tissue culture plate and allowed to adhere for 12 hours in complete DMEM supplemented with 10% FBS, 1X L-Glutamine, and 1X

Penicillin/Streptomycin. After 24 hours, cells were co-transfected with FSTL3 vector and empty eGFP vector at a ratio of 3:1, respectively, or empty eGFP vector alone using

Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s recommended protocol. After 8 hours, growth medium containing transfection reagent and plasmid was replaced with complete DMEM and cells were allowed an additional 24 hours to reach 100% confluence and a minimum of 70% transfection efficiency before inducing differentiation. Differentiation from myoblasts to

myotubes was induced by serum deprivation with basal DMEM supplemented with 2% horse serum.

Treatment of mouse myoblast cells with recombinant FSTL3

FSTL3 was expressed in CHO-S cells (FreeStyle MAX CHO Expression System,

Life Technologies, Grand Island, NY). Briefly, cells were cultured in suspension in

FreeStyle CHO Expression Medium (Invitrogen) at 37°C in 5% CO2. The FSTL3

Human cDNA (Origene Technologies, Cat. SC319496, Rockville, MD) was subcloned into E. coli DH5 alpha competent bacteria (Invitrogen, Cat. 18265-017, Grand Island,

NY) and purified by QIAGEN maxiprep (Qiagen, Valencia, CA). The CHO-S cells were seeded at approximately 3 x 105 cells/mL, and transfected with the FSTL3 plasmid DNA at a cell density of 1 x 106 cells/mL. Protein expression was monitored for one week, and supernatants containing secreted FSTL3 were clarified by centrifugation and concentrated using Millipore Amicon Ultra-15 Filters (10,000 MW cutoff, EMD

Millipore, MA). Concentrated FSTL3 protein was added to C2C12 myoblasts seeded at a density of 7.5 x 104 cells/mL in a12 well culture plate allowed to adhere for 12 hours in complete DMEM supplemented with 10% FBS, 1X L-Glutamine, and 1X

Penicillin/Streptomycin. FSTL3 was added at approximately 0.4 g/mL, 0.04 g/mL, and 0.004 g/mL, since FSTL3 is found in normal serum at approximately 0.04 g/mL.

Differentiation from myoblasts to myotubes was induced by serum deprivation with basal

DMEM supplemented with 2% horse serum after cells had reached 100% confluence.

FSTL3 was added either at the time myoblasts were seeded, immediately before

differentiation, and after differentiation was complete to determine effect of time on treatment. Myotube area was measured to detect possible hypertrophy.

Statistics

Results are reported as the mean ± SEM unless otherwise noted. Statistical analyses were performed using one-way Analysis of Variance (ANOVA) for cell experiments or the Kruskal-Wallis nonparametric test for contractility experiments to model the effects of FSTL3 and FST and exercise on measures of muscle growth and strength using GraphPad software. Sex differences also were considered. Results were considered statistically significant when p < 0.05.

Results

Walking is an insufficient stimulus to increase muscular force in mice

We first examined the stimulated muscular force responses of the WT mice to the walking exercise protocol by subjecting the dissected EDL and SOL muscles to stimulation frequencies from 1-150 hz. The walking exercise protocol used in this study, although sufficient to produce increases in bone growth,30 was not sufficiently rigorous to produce increases in muscle growth and stimulated force in WT mice (Fig. 14, n=10 mice/group). The graph represents the specific force (N/cm2) of EDL and SOL muscles from the male mice in response to stimulation frequencies from 1-150 hz. Similarly, no increases in stimulated force were observed in the male FSTL3 KO mice in response to the walking protocol (Fig. 15, n=5/group). Female mouse muscle showed a similar response, although a lack of sufficient mouse number decreased the confidence in these

results (Figure 16-17, FSTL3 KO No EX n = 3 EDL, 4 SOL; FSTL3 KO EX n = 3 EDL,

4 SOL).

Follistatin family members may perform redundant and non-essential physiologic

functions

We examined the contribution of two follistatin family members on muscle contractile force, and attempted to determine any compensation that might be occurring between the molecules. It was suspected that another member of the follistatin family, circulating FST (FST315), could be compensating for the missing FSTL3 protein in the

FSTL3 KO animals to affect contractile force. To account for this scenario, we had obtained mice lacking FST315 (“FST288”) from Dr. Alan Schneyer from the University of Massachusetts (n=5 FST288 mice/group). We crossed mice lacking FST315 with those lacking FSTL3 to produce double knockout mice (DKO; n=3 DKO male mice/group; n= 5/group DKO EX females and 5 EDL, 4 SOL DKO NonEX females).

We expected that if FSTL3 or FST315 were necessary for full muscle contractile force, we should see differences between baseline stimulated forces in FSTL3 KO or FST288 and WT muscles. We did observe a non-significant difference in baseline force in these animals. Fig. 15 and Fig. 17 shows that EDLs from FSTL3 KO mice tended to be weaker at baseline than the WT mice. We did not observe this difference in the SOLs. We also observed a non-significant difference in the stimulated force of the FST288 mice compared to WT in two instances: the male SOLs and the female EDLs (Fig. 18-19).

Figure 14. Specific Force (N/cm2) of EDL and SOL Muscles from WT Male Mice in

Response to Stimulation Frequencies from 1-150 hz.

The graph shows no differences in stimulated force between the control, non-exercised trained mice and the walking trained mice.

Figure 15. Specific Force (N/cm2) of EDL and SOL Muscles from FSTL3 KO Male

Mice in Response to Stimulation Frequencies from 1-150 hz.

The graph shows no significant differences in stimulated force between the control, non- exercised trained mice and the walking trained mice; however, the FSTL3 KO mice tended to be weaker than the WT mice at baseline.

Figure 16. Specific Force (N/cm2) of EDL and SOL Muscles from WT Female Mice in Response to Stimulation Frequencies from 1-150 hz.

The graph shows no significant differences in stimulated force between the control, non- exercised trained mice and the walking trained mice.

Figure 17. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FSTL3

KO Female Mice in Response to Stimulation Frequencies from 1-150 hz.

The graphs compare the responses of the WT EDL and SOL muscles at baseline and after exercise training to the FSTL3 KO muscles. The graph shows no significant differences in stimulated force between the control, non-exercised trained mice and the walking trained mice in these groups; however, the FSTL3 KO mice tended to be weaker than the

WT mice at baseline.

Figure 18. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FST288

Male Mice in Response to Stimulation Frequencies from 1-150 hz.

The graphs compare the responses of the WT EDL and SOL muscles at baseline to the

FST288 muscles. The graph shows no differences in stimulated force between the EDL muscles, but a trend toward higher forces in the WT SOLs compared to the FST288

SOLs. 69

Figure 19. Specific Force (N/cm2) of EDL and SOL Muscles from WT and FST288

Female Mice in Response to Stimulation Frequencies from 1-150 hz.

The graphs compare the responses of the WT EDL and SOL muscles at baseline to the

FST288 muscles. The graph shows no differences in stimulated force between the soleus muscles, but a trend toward higher forces in the WT EDLs compared to the FST288

EDLs. 70

To determine the effect of compensation, each single genetic knockout was compared to the DKO. For example, if FST315 were compensating for FSTL3, differences between baseline stimulated forces in FSTL3 KO and DKO should be observed for these muscles. Similarly, if FSTL3 were compensating for FST315, differences between baseline stimulated forces in FT288 and DKO should be observed for these muscles. These results were not definitive due to low sample number in the

DKOs, but we observed non-significant differences at higher stimulation frequencies in the EDLs (DKO EDL forces tended to be higher than FST288 and FSTL3KO forces), and a trend toward higher force in the male SOL DKO compared to the FST288. Finally, we would expect to see a difference in forces between the WT and DKO mice since the effects of both FSTL3 and FST315 would be removed. Surprisingly, we observed no significant differences in the stimulated force of the muscles from these two mouse models (Figures 20-22).

Walking does not increase muscle hypertrophy through MSTN inhibition

If walking-upregulated FSTL3 was binding to MSTN, we would expect to observe an increase in muscle hypertrophy in the muscles from the WT animals in response to the exercise, but no corresponding increase in the KO muscles. Muscle weight/body weight ratios were determined for all of the animals tested. We observed no significant increases in muscle hypertrophy in response to walking (Fig. 23) in either the

WT or KO animals.

Figure 20. Specific Force (N/cm2) of EDL and SOL Muscles from FST288, FSTL3

KO, and DKO Male Mice in Response to Stimulation Frequencies from 1-150 hz.

The graphs compare the responses of the FST288 or FSTL3 KO EDL and SOL muscles at baseline to the DKO muscles. The graphs shows no differences in stimulated force between the FSTL3 KO and DKO SOL muscles, but a trend toward higher forces in the

DKO muscles in all other comparisons.

Figure 21. Specific Force (N/cm2) of EDL and SOL Muscles from FST288, FSTL3

KO, and DKO Female Mice in Response to Stimulation Frequencies from 1-150 hz.

The graphs compare the responses of the FST288 or FSTL3 KO EDL and SOL muscles at baseline to the DKO muscles. The graphs shows no differences in stimulated force between the SOL muscles, but a trend toward higher forces in the DKO EDLs compared to the FST288 and FSTL3 KO EDLs.

Figure 22. Specific Force (N/cm2) of EDL and SOL Muscles from WT and DKO

Mice in Response to Stimulation Frequencies from 1-150 hz.

Muscles from female mice are on the left and from males on the right. The graphs compare the responses of the WT EDL and SOL muscles at baseline to the DKO muscles. No differences in stimulated force were observed between WT and DKO muscles.

Figure 23. Muscle Weight to Body Weight Ratios from Male and Female FSTL3 KO

Mice Compared to WT Mice.

The graphs show comparisons between the EDL and SOL muscles from both groups with or without exercise training. No significant differences in the MW/BW ratios were observed.

Loss of FSTL3 does not affect muscle fatigability

We next wanted to examine whether loss of FSTL3 would affect the fatigability of the muscles. Fatiguability is defined here as the loss of muscular force over time with repeated stimulations. While we did not test for change in muscle fiber type directly, this test could provide some clue as to potential fiber responses and gene expression changes that could occur with MSTN inhibition. Oxidative (“slow twitch”) fibers tend to have smaller cross sectiona area (CSA), higher capillary density, lower force levels, and high fatigue threshold, while glycolytic (“fast twitch”) muscles have higher CSA, lower capillary density, higher forces, and low fatigure threshold.186 MSTN inhibition may result in a shift to the glycolytic phenotype.187 We hypothesized that FSTL3 KO muscles, in which MSTN was unopposed, may more closely resemble the oxidative, slow-twitch phenotype. During this test, muscles were fatigued at ½ Tmax for 5 min, with stimulation interval of 2 s and 25% duty cycle.183 Instead, we observed that walking exercise had no effect of the fatigability of the muscles in either the WT or KO animals

(Fig. 24). Similar results were obtained for the females, and for FST288 and DKO groups, and no animals showed significant MW/BW ratios at baseline (data not shown).

Figure 24. Representative Graph of Comparison of Percent Fatigue between FSTL3

KO and WT Muscles.

The graph shows the response to the fatigue test of male EDL and SOL muscles from either FSTL3 KO or WT mice at baseline and after exercise training. Muscles were fatigued at ½ Tmax for 5 minutes. No significant differences between groups were observed. Graph is representative of male and female mice from the FST288 and DKO genotypes.

FSTL3 overexpression does not produce myotube hypertrophy

Although the ex vivo experiments were limited by the ability of the walking protocol to produce an acceptable hypertrophic stimulus, we still wanted to determine the effect of FSTL3 on muscle at the cellular level. Thus, we transiently transfected mouse myoblast C2C12 cells with a plasmid containing FSTL3 and GFP, or a plasmid containing the GFP plasmid alone and measured the size of the resulting myotubes (Fig.

25). We expected that the FSTL3 over-expressing myotubes would be larger in size than the GFP expressing control myotubes. Instead we observed no significant differences in the size of the myotubes in either group (Fig.26).

Addition of extracellular recombinant FSTL3 does not produce myotube hypertrophy

Since FSTL3 is a circulating mediator in vivo, we attempted to decipher the effects of FSTL3 added exogenously in cell culture. The FSTL3 protein (Figure 27) was expressed in CHO cells and then added separately to C2C12 cells at three time points during the growth of the cells: during initial plating, immediately before serum starvation to initiate differentiation into myotubes, or after differentiation into myotubes had occurred (Fig. 27). The time-dependent development of myotubes was confirmed in cellular extracts by Western blot probing for differentiated myotube specific markers, mitsugumin 53 (MG53) and desmin188, 189 (Figure 28). Once again, myotube sizes were measured. No significant differences in myotube size in response to the FSTL3 treatment were observed at any point in time (Fig. 29).

Figure 25. Experimental Design for Transfection of Mouse Myoblast C2C12 Cells with Fstl3.

C2C12 myoblasts were co-transfected with a plasmid vector containing Fstl3 or Gfp.

Differentiation to myotubes was induced by replacement of complete medium with 2% horse serum. The pictures show representative GFP-transfected myoblasts and FSTL3- transfected myotubes.

Transfection Results 100

) 2 80

Myotube Size (umSize Myotube

FSTL3 transfected

CON (GFP) transfected Group

Figure 26. Results of the Transfection of Mouse Myoblast C2C12 Cells with Fstl3

Experiment.

Myotube size in response to FSTL3 overexpression was measured. No significant differences were observed in the myotube sizes between the FSTL3 transfected and the

GFP transfected cells.

Figure 27. Experimental Design for the Addition of Recombinant FSTL3 to Mouse

Myoblasts and Myotubes Experiment.

FSTL3 protein was expressed in CHO cells, and then added separately to cells at three different time points during the growth of the cells: during initial plating of the myoblasts, immediately before serum starvation, and after differentiation into myotubes had occurred. The Western blot is the concentrated FSTL3 protein expressed from CHO cells.

Figure 28. Confirmation of Myotube Development in Cellular Extracts from the

Recombinant FSTL3 Treatment Experiment.

Results of Western blotting showing time-dependent development of myotubes after

C2C12 myoblasts were treated with recombinant FSTL3. The blots were probed for the differentiation-specific markers, mitsugumin 53 and desmin.

Figure 29. Myotube Size in Response to Recombinant FSTL3 Treatment.

FSTL3 protein was expressed in CHO cells, and then added separately to cells at three different time points during the growth of the cells: during initial plating of the myoblasts

(1), immediately before serum starvation (2), and after differentiation into myotubes had occurred (2). FSTL3 was also added at differing concentrations (µg/mL). No significant differences in myotube size were observed in response to the FSTL3 treatment at any point in time or at any concentration of FSTL3 in comparison to the control condition.

Discussion

Mechanisms of exercise mediated bone and muscle coordination remain largely undetermined. Through this study, we attempted to determine the effects of the FSTL3

protein, a known regulator of bone growth that is uregulated by walking exercise, on muscle strength through its actions on MSTN. FSTL3 is a mechanoresponsive protein that is upregulated in response to walking exercise.30 FSTL3 is known to bind to MSTN and prevent its downstream actions that could result in decreased muscular hypertrophy and increased muscular atrophy. However, we did not observe an effect of the loss of

FSTL3 on the muscular strength of our animals (Figures 15 and 17).

One reason that we did not observe differences in FSTL3 KO versus WT animals was the lack of a protocol sufficient for increasing muscular force in the mice (Figures 14 and 16). We were unable to produce a training effect on the WT mice using our walking protocol; however, the difficulty in increasing muscle mass in mice through exercise training has been documented.190 Interestingly, this protocol was sufficient for producing an increase in bone growth in the WT mice. Our results indicate that serum FSTL3 upregulation, which has been observed in response to short-term walking exercise30 may not directly influence a change in muscular hypertrophy; however, the effects of other exercise protocols on mouse FSTL3 levels should be examined. Further, future analyses using serum collected during the current study will be tested to assess if an upregulation in FSTL3 had occurred in response to the six weeks of walking exercise. It is possible that a resistance or high intensity exercise training protocol could have greater effects on the FSTL3/MSTN system than we were able to detect here, as resistance protocols have been shown to affect this system in humans.86, 177 To our knowledge, this is the first study to determine the effects of low-intensity walking exercise on FSTL3 and MSTN in mice.

Although all MSTN mediators were not tested, we did attempt to address the issue of whether FST could be compensating for the loss of FSTL3 by using multiple genetic knockouts in our contractility experiments. Although FST also is upregulated in response to exercise, its global knockout is lethal in the mouse. Such a molecule could not be further developed as a MSTN inhibitor/treatment for bone and muscle strength, as it serves essential functions in the body that could be affected by overexpression. FST is upregulated by exercise,177 stimulates myogenic transcription factors in myoblasts, blocks

MSTN, activin, and TGF-β1,71 and potentially could compensate for deleted FSTL3 in binding MSTN. This possibility is unlikely as the literature consistently depicts dissimilar roles for FSTL3 and FST in the circulation, binding partners, structure, etc.62,

31, 63 Regulation of MSTN is believed to occur at the tissue level through autocrine/paracrine levels rather than through the circulation62 and circulating FST is primarily bound to activin.31 However, we did include an analysis of FST levels in these studies to determine if an upregulation does occur in response to FSTL3 loss. Our results indicated that FSTL3 may be compensating for FST315 and that FST288 may be compensating for FSTL3, as non-significant differences between baseline stimulated forces in FST288 or FSTL3KO and DKO were observed for these muscles (Figures 20 and 21). These results were not definitive, as the sample size for the DKO mice was smaller (n=3) than the other groups, decreasing confidence in these results. While these results are equivocal and could be due to the small sample size in the DKOs, future testing will help to clarify the roles of these mediators on MSTN. For example, mouse serum collected in this study should be assayed for differences in serum upregulation of

the mediators described here. FSTL3 KO mouse serum may show an upregulation in

FST levels, whereas FST288 mice may produce an upregulation in serum FSTL3 to compensate for reduced levels of the missing mediator. Serum from all of the mouse models should be assayed for MSTN levels. Finally, the muscle tissues collected during this study should be analyzed for protein levels of these mediators to determine if compensation and interactions between FSTL3/FST and MSTN is occurring at the tissue level.

The similar results in stimulated contractile forces between DKO and WT mouse muscles at baseline is interesting (Figure 22). These results indicate that the two follistatin family molecules we have studied here may not be essential circulating mediators for muscle force production. These results were further supported by the lack of significant difference between either KO model and the WT muscles (Figures 15, 17,

18, and 19). We did observe a non-significant difference in baseline force in these animals. Figures 15 and 17 show that EDLs from FSTL3 KO mice tended to be weaker at baseline than the WT mice. We did not observe this difference in the SOLs. We also observed a non-significant difference in the stimulated force of the FST288 mice compared to WT in two instances, the male SOLs and the female EDLs (Figures 18 and

19).

The fact that the MW/BW ratios were not significantly different from WT in all groups further indicates a lack of MSTN involvement at baseline (Figure 23). It appeared that there may be a differential response of FSTL3 treatment by muscle type in the female; that is, the fast twitch EDL muscles are more sensitive to the loss of FSTL3 than

the slow twitch SOL muscles (Figure 17). Although this trend was not consistent or significant, this finding bears further investigation, as a change in fiber type from slow to fast in MSTN-null mice has been reported,191 and MSTN is preferentially expressed in fast-twitch muscle fibers.192

It has been shown previously that regulation of MSTN to regulate glucose homeostasis by these FST family genetic models differs depending on tissue and primary role.62 It should be noted that other FST family molecules could be binding to MSTN to affect muscle growth and strength, but the focus of these studies was restricted to FSTL3.

For example, we did not test for FSTL1, although this molecule also is upregulated in response to exercise.193 However, the roles of FSTL1 have been most studied and well characterized in the heart.194, 195, 196, 197 Although blockade of TGF-β family members other than MSTN can influence muscle size, these molecules, including TGF-β1, GDF-

11, activins, BMP-2, and BMP-7, play other primary roles in the body that could be affected by their inhibition.198, 199 The normal appearance, function, and fertility of our

DKO mice suggested that these molecules were not being affected. However, future studies could quantitite these other molecules and MSTN regulators including MSTN propeptide, SMURF1, and GASP-1. However, it should be noted that methods to quantify MSTN cannot distinguish between active and latent states, therefore abundance does not necessarily correspond to activity.178 Another limitation of this study is that we did not test for ActRIIB levels. Any downregulation in the expression of this receptor could account for decreases in MSTN activity, and decreases in response to exercise has been observed.200

We also did not observe an increase in myotube hypertrophy in response to

FSTL3 overexpression or recombinant FSTL3 treatment of C2C12 cells. While these experiments may show a true effect of FSTL3 on C2C12 cells, it is also possible that the cell culture system used was already fully saturated with circulating FSTL3 from serum in the cell culture media. Any additional FSTL3 added to this system may be redundant for binding and inhibition of MSTN. To confirm a role of FSTL3 in mechanoresponses, better gain-of-function approaches would be ideal. Unfortunately, transgenic mice overexpressing FSTL3 are sterile and cannot be propagated.201 One other study examined FST treatment to inhibit MSTN in C2C12 cells.71 This study found that recombinant MSTN and activin A can inhibit differentiation ability of C2C12 cells, while

FST can allow the myoblasts to undergo differentiation in both cases. FST also was shown to reduce expression and phosphorylation of SMAD2.

It has been reported that myoblasts lacking MSTN proliferate faster but differentiate into myotubes more slowly.94 Future experiments involving treatment with exogenous recombinant FSTL3 should include an assessment of number of myotubes formed over time to address the possibility of this occurrence with MSTN inhibition.

Addition of FSTL3 may result in slower myotube differentiation through MSTN inhibition, although another mechanism is possible. Metalloproteinase activity may be needed for cell fusion during myogenesis.176 Confirmed FSTL3 interaction with one such metalloproteinase, ADAM12, has been shown to mask the site needed by ADAM12 to drive cell fusion in osteoclasts.176 Such a mechanism may be at work in the case of myogenesis as well.

FSTL3 has been shown previously to be upregulated in the serum of mice, rats, and humans in response to walking exercise.30 The most significant response was observed after 7 days of walking, and then a decrease was observed at 15 days, although levels were still significantly elevated above baseline. For this reason, we believed that

FSTL3 showed promise as a circulating mediator that could integrate bone and muscle communication. However, we determined that walking exercise is not sufficient for increasing muscle growth and strength in the mouse, in spite of the observed increase in bone growth. Future studies are needed to determine the promise of FSTL3 for bone and muscle growth in humans. While walking exercise is commonly prescribed for individuals with osteopenia or osteoporosis, and it is known to be effective at increasing bone mass, the effectiveness of a walking exercise prescription alone may be a questionable practice. Exercise of varying intensities and impact, such as resistance training or high-intensity impact training, may be more beneficial for increasing both bone and muscle mass. The effect of altering exercise modes, intensities, and/or durations on serum FSTL3 should be studied in both mice and in humans.

It is possible that FSTL3 is regulated so that a serum increase would not necessarily lead to changes at the cellular level (e.g., receptor downregulation/binding proteins, etc.). For this reason, changes in FSTL3 at the cellular level should be assessed.

Additionally, changes in expression of regulators of FSTL3, including activin and Smad proteins, should be analyzed in bone and tissues.202, 60

Studies show conflicting results regarding the effectiveness of MSTN inhibition to increase muscle growth and strength. Heterogeneity of studies in terms of animal

models used, time course of treatment and/or sampling, and treatment strength all complicate interpretation of experimental results and make comparisons between studies difficult.72 Differences in gene expression in contractile proteins have been observed according to whether animals were MSTN null versus inhibited at the adult stage.187

Differences in results also are observed when examining MSTN inhibition by different regulators. For example, a cDNA containing FST344 was delivered by adeno-associated viral vector into the muscle to produce a gene product capable of producing the FST315 circulating form.182 Increased muscle size and strength were observed in mice and in monkeys using this approach. Further, since FST315 has reduced affinity for heparin, the gonadal-pituitary axis known to be affected by the follistatins was less likely to be altered. The results of MSTN inhibition using FST315 also was compared to inhibition using GASP-1 and FSTL3. Significant changes in muscle weights and hindlimb grip strength were only observed with the FST315 treatment. This study showed that while

MSTN inhibition may be a promising therapeutic option for increasing muscle growth,

MSTN inhibition through FSTL3 may not be the most effective option. The literature is conflicting with regard to MSTN inhibition and the changes in muscle fiber size and/or number, and differences observed may be a factor of species differences or extent of myostatin inhibition.76 Future studies should further clarify the effect of MSTN inhibition on fiber type switching.203, 204, 205

It is clear that much is still unknown about the most effective way to inhibit

MSTN to produce the best gains in muscle. Interestingly, another study using soluble

ActRIIB to inhibit MSTN showed that inhibition was minor in the normal, healthy adult,

and may be most effective in the cases of protein degradation or increased MSTN levels.67 The study also cited work indicating that unexpected increases in ubiquitin ligase transcripts have been observed with MSTN blocking, and that there is a specific timecourse to involvement of the signaling pathways involved due to MSTN inhibition.

Observed decreases in capillary density per muscle fiber area needs further study to determine its effects on muscle function.

Another study investigated the effects of MSTN inhibition alone as well as in combination with exercise.206 Mice were treated with siRNA directed against MSTN and/or were progressively treadmill trained, but the two treatments did not have a synergistic effect on muscle growth. Short interfering (siRNA) treatment produced a greater effect than exercise alone, which did not result in an increase in muscle mass.

The mice were running at 15 m/min in this study; as our mice we walking at 8 m/min, we expected to see similar results in terms of muscle mass. Of further interest is the observation that MSTN knockdown in these mice resulted in an increase in FST mRNA in muscle, showing that MSTN may be regulating expression of its own antagonist.

Exercise did not influence expression of FST, but prevented the increase in FST when combined with siRNA knockdown. Serum FST decreased as a result of the siRNA knockdown and exercise independently, but increased when knockdown and exercise were combined. These results indicate that MSTN inhibition through exercise alone may be complicated. The study also observed that sex dependent effects have been noted with

MSTN inhibition.

Yet another study by Matsakas et al. described that hyertrophic muscle as a result of MSTN inhibition does not lead to proportional changes in force, and that endurance exercise specifically might normalize the hypermuscularity effects.186 MSTN null mice subjected to running or swimming showed reduced muscle fiber size, increased muscle oxidative properties, increased capillary density and improved muscle force. This study again demonstrated that effects of MSTN deletion are modifiable by exercise.

The increased oxidative properties observed by Matsakas et al. is interesting because reports of decreased oxidative capacity have also been shown to occur after blocking of MSTN and activins by souble ActRIIB-Fc. In one particular study, evidence of decreased oxidative pathway expression was determined using microarray analysis.187

These results are important as they show that inhibition of MSTN, even when effective in producing an increase in muscular size, could result in potentially undesireable consequences in terms of the function of the muscles. However, another study shows that this phenotypic change in oxidative capability may only occur as a result of MSTN null versus MSTN inhibition in the adult.207 Changes in myosin heavy chain isoforms and succinate dehydrogenase activity were observed in MSTN null mice. In spite of larger muscle mass in the MSTN null animals, these muscles have in fact been observed to be weaker than WT muscles and have more Type IIb fibers that contained tubular aggregates and reduced mitochondria.205

In spite of its challenges, MSTN has been regarded as an attractive target for the development of therapeutics to increase muscle growth through its inhibition. Tsuchida et al., reported in a review that MSTN inhibition can be effective in increasing muscle

mass and force.208 For example, monoclonal antibodies to MSTN or a soluble MSTN propeptide have been effective in mdx mice. An adeno-associated virus (AAV) delivery system for the MSTN propetide improved muscle mass and force in a limb girdle muscular dystrophy model. Soluble ActRIIB injections or a dominant negative ActRIIB resulted in up to a 60% increase in muscle mass in mice. Other studies have shown success with MSTN inhibition to increase muscle mass in the mouse;209 however, when these mice are examined closely, muscles have decreased capillary density and angiogenic signaling.67 Off-target effects in humans also have been observed. A single dose of soluble ActIIRB in postmenopausal women caused suppression of FSH, nose and gum bleeding, and dilated vessels in the skin in healthy volunteers.178

In terms of methods to increase muscle mass based on FST in particular, a mutant

FST molecule with reduced activin binding activity transgenically expressed in mdx mice increased muscle mass.76, 203 Interestingly, this treatment also decreased adipose tissue in these mice.63 An alternatively spliced cDNA encoding the product FST315 was delivered by AAV directly to muscle in order to bypass off target effects to the reproductive system.182, 181 This approach was successful in increasing muscle size and strength in mice in non-human primates. Interestingly, delivery of genes encoding

FSTL3 and GASP-1 also were included in this study, and while GASP-1 expression significantly increased muscle weight, neither protein increased muscle force. Finally, this particular FST construct was used in a human proof of principle clinical trial against

Becker muscular dystrophy with mixed success, but no adverse outcomes.210

Conclusions

This study provides information on the effectiveness of walking exercise in the mouse to promote bone and muscle cross-talk through the MSTN/FSTL3 mediators.

Circulating mediators are becoming increasingly studied in order to determine how muscle and bone communicate. While we did not find a significant role for the FSTL3 protein in mediating muscle hypetrophy through MSTN inhibition using our transgenic mouse model and cell culture approaches, it is clear that additional experiments to determine molecular mechanisms of exercise are needed. While FSTL3 is a mechanoresponsive protein that regulates bone health in response to low intensity, low impact walking execise, the use of walking to upregulate FSTL3 to neutralize MSTN is insufficient for increasing muscle force and hypertrophy. Future studies should determine a more effective exercise protocol for improving bone and muscle health, likely requiring the use of eccentric exercise to complement aerobic walking exercise.

Discovery and characterization of molecular mechanisms mediating this process could allow for effective exercise prescriptions to increase musculoskeletal health.

Chapter 4: The Role of Exercise and the Follistatin Mediator in Cardiac Health

Introduction

The significance of heart failure in the U.S.

An estimated 5.1 million Americans over the age of 20 have heart failure, and by

2030, the prevalence of heart failure is expected to increase 25% from 2013 estimates.211

The five year survival rate for heart failure is approximiately 50%, and the economic impact of this disease is estimated to be $100 billion per year.212 Heart failure is characterized by initial compensatory changes in physiology and structure that maintain heart function, including the myocyte hypertrophy, chamber dilation, and matrix remodeling that comprises left ventricular hypertrophy,213 but then by progressive dysfunction that results in end stage heart failure and mortality.214 Despite the magnitude of this health problem and the multiple studies addressing it, molecular mechanisms of heart failure are likely complex and remain unclear. Recently, the roles of secreted factors in the heart that could regulate pathological hypertrophy, including follistatin

(FST) and related molecules, have been characterized.215, 216, 197 Given the role of FST in response to exercise as summarized in previous chapters, and the link between exercise and cardiovascular health, the aim of this study was to determine the effects of follistatin family molecules on heart function in mice.

Exercise is beneficial for cardiovascular health

It is well known that exercise is beneficial for maintenance of cardiovascular health217 and prevention of further pathology in those with established cardiovascular disease218, 219 or in those with cardiovascular risk factors.220, 221 Exercise has been shown to be beneficial in reducing the infarct size and pathology associated with myocardial infarction (MI).222 In a study comparing the effects of exercise training in mice before and after MI, mice were exercised by voluntary wheel running for two weeks prior to inducing MI.223 After MI, one group of mice ran for eight weeks, while the other group remained sedentary. Survival was improved in the group that had been exercise trained before MI, while the other exercise groups showed attenuated fibrosis, apoptosis, left ventricular dysfunction, and pulmonary congestion compared to sedentary control mice.

Separate studies showed that exercise may mediate survival via molecular mechanisms such as upregulation of vascular endothelial growth factor (VEGF), leading to increased angiogenesis,224 improvements in calcium handling and myofilament function,225, 226 and increased endothelial nitric oxide synthase (eNOS) expression.227, 228 Other molecular mediators also are being studied, and these studies suggest that the beneficial effects of exercise on the heart are likely multifactorial.229, 230, 231 Here we have characterized the effects of one such mediator, follistatin-315 (FST315), on the failing heart.

Background on structure and functions of follistatin

FST, a molecule that blocks secretion of follicle-stimulating hormone from the pituitary,44 binds and regulates members of the transforming growth factor beta (TGF-β) family including activins, myostatin (MSTN) and bone morphogenetic proteins

(BMPs).33 Follistatin contains three conserved FST domains, whereas the follistatin-like

(FSTL) proteins contain at least one follistatin domain.232 These molecules, originally believed to act as endocrine regulators of reproduction,233, 201 are now known to also act in autocrine/paracrine fashion234 to influence numerous physiological processes including liver homeostasis, wound repair, and response to inflammatory stimuli based on their distinct structures and expression patterns.235, 33 Follistatin knockout in mice is neonatal lethal.47 These mice display retarded growth as well as multiple skeletal, skin, and muscle defects, indicating that FST is a crucial modulator of TGF-β family members during development and throughout life for normal tissue function.52, 54, 53 FST is produced in many tissues including the gonads, skin, kidney, bone, pituitary, and heart.235, 236

The gene encoding FST produces three isoforms that differ in biological activities and cell surface binding capabilities (Figure 2).237 The FST315 isoform contains all six exons, and proteolytic cleavage of the FST315 C-terminal tail results in production of

FST303.33 Lack of exon 6, which codes for the acidic C-terminal tail of the putative full- length protein, results in FST288.33 The missing acidic C-terminal tail region found in soluble FST315 allows FST288 to bind cell surface heparin-sulfated proteoglycans. This difference in cell surface binding properties accounts for the differential actions of these molecules.33

Actions of follistatin and the follistatin-like proteins in the heart

The follistatins and follistatin-like proteins play various roles in the heart. FST,

FSTL1, and FSTL3 are known to affect heart development either directly or through their

modulatory actions on the TGF-β family members, with FST localized to the endocardium in developing heart valves.238 The follistatin-like molecules, including

FSTL1 and FSTL3, appear to have differential actions in the heart. FSTL1 expressed in cardiac myocytes functions in an autocrine/paracrine manner to antagonize myocyte hypertrophy,197 while FSTL3 appears to antagonize the role of activin in response to injury.239 In heart failure, FSTL1 is believed to be protective, and may modulate myocardial inflammation.194 Serum and myocardial FSTL1 has been shown to be elevated in heart failure,197 where its levels significantly correlate with brain natriuretic peptide (BNP) levels. Overexpression of FSTL1 promotes revascularization and endothelial cell migration, while it suppresses apoptosis, proinflammatory cytokine expression, and proteins involved in extracellular matrix degradation, including MMP-1 and MMP-3.194 Cardiac specific FSTL1 knockout mice subjected to transverse aortic constriction (TAC) displayed hypertrophic hearts as well as an increase in septal and left ventricular wall thickness and reduced fractional shortening when compared to control mice.197 Further, myocyte hypertrophy and fibrosis and BNP and atrial natriuretic factor

(ANF) gene expression were increased in FSTL1 KO mice.197 Administration of exogenous FSTL1 is protective against ischemia-reperfusion injury.197 In addition, the cardioprotective effects of FSTL1 are not limited to myocardial FSTL1, as muscle derived FSTL1 attenuates neointimal formation in response to arterial injury.229 This muscle derived FSTL1 may be released in response to exercise training, thus partly explaining the potentially therapeutic effect of exercise on cardiovascular health.229

FSTL1 appears to function independently of TGF- proteins, as the Smad2 signaling

pathway was not activated.197 In terms of downstream signaling pathways, AMP- activated protein kinase (AMPK) signaling is thought to prevent cardiac hypertrophy,240,

241 and phosphorylation of the Thr172 residue of the AMPK  subunit as well as downstream endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser1179 were reduced in FSTL1 KO mice. These effects were not observed in an FSTL1 overexpression model. Interestingly, neither heart size nor function was affected in either mouse model during baseline conditions; it appeared that FSTL1 functions in conditions of pressure overload induced hypertrophy.197

While previous studies illustrate the function of FSTL1 in the heart, less described is the role of FSTL3. FSTL3 is thought to play a major paracrine role in the regulation of fibrosis in response to pressure overload in the heart, as it promotes fibroblast proliferation and adhesion as well as collagen production in fibroblasts.242 Wild-type

(WT) mice showed a strong increase in the heart failure markers ANP and BNP and upregulation of genes related to cell adhesion, extracellular matrix (ECM), and cytoskeleton 21 days after TAC, whereas these markers were significantly decreased in

FSTL3 KO mice.242 FSTL3 expression has been shown to be necessary for full development of cardiac fibrosis and hypertrophy.242, 215

In contrast to FSTL3, activin, a TGF-β family member to which FST and FSTL3 bind, is believed to have a protective role following MI, but conflicting evidence also has been reported. Serum levels of activin A are elevated in heart failure patients and correlate with disease severity,243 and in a rat experimental hert failure model, activin mRNA and protein expression was significantly increased in ischemic cardiomyocytes.243

Administration of exogenous activin A on neonatal rat cardiomyoctes significantly increased the expression of biochemical mediators that affect cardiac remodeling and healing (i.e., ANP, BNP, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-

β1, and monocyte chemoattractant protein (MCP)-1).243 Activin inhibits myocyte hypertrophy and also has been shown to decrease expression of hypertrophic markers such as BNP and ANF.215 On the other hand, increased activin after MI was shown to increase fibrosis and collagen deposition in the damaged heart, but this study appeared only to establish an association versus cause and effect.236 Another study suggests that activin may lead to heart failure by suppressing growth hormone.244 It is evident that activin A plays a role in heart failure, but the precise role of this mediator in pathogenesis and healing after MI requires further study. FSTL3 and FST neutralize activin’s actions.242 FSTL3 bound to activin A can modulate the heart’s response to ischemic stress239 and pressure overload.215 In this study, systemic overexpression of activin A protected hearts from ischemia/reperfusion injury, while FSTL3 treatment abolished the protective effects of activin A in cultured myocytes. A balance between these two molecules may be an important regulatory system in the response of the heart to ischemic insults. Interestingly, exogenous administration of FST has been shown to reduce ischemia/reperfusion injury in mice, while activin A exacerbated these effects in cardiomyocyte cell culture.245 MSTN, the other major TGF-β family member regulated by FSTL3 and FST, was originally believed to be skeletal muscle specific, but has been shown to be upregulated in the heart after MI and modulates AKT activity.246, 247, 248

While upregulation of MSTN expression in heart failure is established, the specific role

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that MSTN is playing in the pathology is not described. Cardiac upregulated MSTN may be downregulating skeletal muscle hypertrophy as well as cardiac hypertrophy,249 and increased MSTN in the heart has been shown to lead to interstitial fibrosis.250 However,

MSTN ablation specifically in adult cardiomyocyes also has been shown to lead to heart failure,251 indicating that a regulated amount of this mediator is likely required by the heart for normal function.

A 2008 study determined the expression levels and explored the roles of the FST family and related members in heart failure.216 Myocardial samples from healthy human donors and those with end-stage heart failure at the time of implantation of left- ventricular assist devices (LVAD) were examined. FSTL1 and FSTL3 expression was elevated in heart failure, but FST and activin expression showed no difference between the two groups. MSTN could not be reliably detected by PCR in either group. In a TAC model, FSTL1, FSTL3, and FST were found to be induced. FSTL3 correlated with heart failure markers BNP and α-actin, whereas FSTL1 correlated negatively with α-actin.

Higher FSTL1 levels at the time of LVAD implantation corresponded with better outcomes, wherease the opposite was true of FSTL3. Interestingly, microarray data analysis of gene expression patterns corresponding to high FSTL1 levels was similar to patterns correlating to high FST levels, suggesting that these two mediators may be involved in similar processes in the heart. While the activities of these molecules in the heart have been somewhat characterized, the role of the FST315 molecule in the heart, to our knowledge, has not been determined.236 Further, sex specific differences in response

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to these hormones have not been characterized. Finally, the effects of exercise on these molecules and their subsequent effect on the heart have not been described.

In this study, we used genetically modified mice that express only the FST288 isoform, mice lacking the Fstl3 gene, and mice lacking both FST315 and FSTL3 to test the roles of FST315 and FSTL3 in the adult heart. Since these are unique molecules in structure and could have different functions in the heart, we decided to focus our research on the least characterized molecules, FST288 and FST315. Exercise training in female

FST288 mice blunted the trend toward a hypertrophic response that was observed only in exercised male FST288 mice, and systolic and diastolic left-ventricular internal dimensions (LVIDs/LVIDd) were maintained at WT levels in the females in response to exercise. Although all hearts appeared to maintain normal function, exercise in the male mice elevated LVID. While loss of FST315 expression has limited effects on the heart at the resting state, when these mice are subjected to pressure overload through transverse aortic constriction (TAC) surgery, they appear to be resistant to the compensatory cardiac hypertrophy present in wild type mice by 4 weeks post surgery. Both cardiac structure and function (as measured by echocardiography) following TAC are improved in the

FST288 mice compared to the WT mice. This response is likely due to modification of the AKT signaling pathway. Overall, our data illustrate that FST315 is an important contributor to the progression of pressure overload induced cardiac hypertrophy.

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Methods

Mouse models

All experiments conformed to the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health, and were approved by the

Institutional Animal Care and Use Committee of The Ohio State University. Male wild- type mice (The Jackson Laboratory), age 12 weeks, were housed at constant temperature

(22 ± 2˚C) and provided with a 12-h light/dark cycle. Mice were given standard lab chow and water ad libitum. FST288 mice were developed and kindly provided by Dr.

Alan Schneyer, and creation and characterization of these mice has been previously described.48 These mice resulted from a knock-in mutation was made on a mixed

129S4/SvJae × C57BL/6 background. Other transgenic mouse models compared to the

C57BL/6 WT included a previously described Fstl3-/- mouse model (“FSTL3 KO,”

C57BL/6 background)31 and FSTL3/FST288 double knockout (DKO) mice.

Exercise protocol

Mice were subjected to treadmill walking on a small animal treadmill (Columbus

Instruments, OH) at a speed of 8 m/min for 45 minutes/day for 6 weeks. Mice were euthanized one day after the last exercise regimen, and hearts were harvested for analysis.

Mice were randomly assigned to exercise versus non-exercise (CON) groups (n=5 mice/group).

Transverse aortic constriction (TAC) and echocardiography

Mice were initially anesthetized with inhaled 3% isoflurane and then intubated with a 20G intravenous catheter and ventilated with a mixture of O2 (0.3 L/min) and 1.5-

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2% isofluorane (tidal volume 250 μL, 120 breaths/min) with a mouse respirator (Harvard

Apparatus, Holliston, MA). Animals were placed in a supine position, and the body temperature was monitored with a rectal probe to maintain temperature at 36-37C using a heating pad. A partial thoracotomy was then performed through the second intercostal space, and 6-0 silk ligature was used to tie two knots around the transverse aorta after separation of tissue from the thymus arch.

Mice were anesthetized using isofluorane and echocardiography was performed using a VisualSonics Vevo770 with 30 MHz probe. Measurements were taken in M- mode for each mouse. Doppler echocardiography was used to determine structural and functional measurements of mouse hearts subjected to TAC.

Western blotting

Heart tissue samples were homogenized in radioimmunoprecipitation assay

(RIPA) lysis buffer (Cell Signaling Technology) with a protease inhibitor mixture

(Roche) and phosphatase inhibitor mixture (Sigma). Protein content was determined by the Bradford method. Equal amounts of protein (40 μg) were separated in denaturing

SDS 8–12% polyacrylamide gels and transferred to nitrocellulose membranes.

Membranes were immunoblotted with the primary antibodies at a 1:1,000–5,000 dilution followed by incubation with the secondary antibody conjugated with HRP at a 1:1,000–

10,000 dilution. Proteins were visualized using ECL Western Blotting Detection kit

(Amersham Pharmacia Biotech). Membranes were also analyzed using appropriate fluorescent secondary antibodies including IRDye 680 or IRDye 800 conjugated goat

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anti-rabbit or goat anti-mouse IgG (LI-COR, NE). Blots were imaged and quantitatively analyzed by LI-COR Odyssey imager and Odyssey application software, version 2.1

Statistical analysis

Data are expressed as mean ± SEM unless otherwise indicated. The results of experiments were analyzed by unpaired t-test using Graphpad Prism v4.0 (GraphPad

Software, San Diego, CA). In all cases, a p value of < 0.05 was considered statistically significant.

Results

FSTL3 KO hearts were examined for function in response to exercise training.

These hearts were determined to have normal weight (Figure 30), ejection fractions (EF) and fractional shortening (FS, Figure 31) (measures of how much blood is pumped out of the left ventricle and a measure of the pump function of the heart, respectively), and left ventricular end-diastolic and end-systolic internal dimensions (LVID, a measure of stroke volume, Figure 32) compared to the control WT mice. Only FSTL3 KO exercised animals had a decreased LVID in comparison to WT exercised mice; however, this could be the result of random variation. DKO mice also had normal heart function at baseline

(Figure 33), as indicated by normal LVID measurements with or without exercise.

Analysis of the effects of FST on the adult heart are complicated due to the neonatal lethality of the FST global knockout. We suspected that such lethality indicated the importance of this molecule on normal mouse development.47 We also had access to the unique FST288 mouse model to test the cardiac role of FST315, thus we examined the FST288 mice more closely.

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Weights of male and female FST288 hearts showed possible differential response to exercise training (Figure 34). The heart weight to body weight ratios for the male

FST288 exercised animals was significantly higher than the female FST288 exercised animals. The same result was observed for the heart weight/tibia length measurements.

Although these heart weights were not significantly different from their respective male and female control animals, a trend toward increasing heart weights was observed for the exercised male animals in comparison to the control male animals, whereas the female mice showed no change from baseline.

Male and female FST288 hearts show potential differential remodeling in response to exercise training (Figure 35). Echocardiograph analysis of LVID indicated that LVIDs and LVIDd from exercised male FST288 hearts were significantly elevated when compared to exercised female mouse hearts. This response in the males potentially indicates an increase in physiologic hypertrophy, while female mice responded to exercise training with a non-significant decrease in LVID.

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Figure 30. Heart Weight to Body Weight (HW/BW) and Heart Weight to Tibia

Length (HW/TL) Ratios for FSTL3 KO Hearts Compared to WT Hearts.

Hearts were examined for changes in weight in response to exercise training, sex, and genotype. No significant differences were observed between groups. Figure produced in collaboration with Dr. Zhaobin Xu. 107

Figure 31. Ejection Fraction and Fractional Shortening Measurements for FSTL3

KO Hearts Compared to WT Hearts.

Hearts were examined for changes in these measurements in response to exercise training, sex, and genotype. No significant differences were observed between groups.

Figure produced in collaboration with Dr. Zhaobin Xu.

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Figure 32. Left Ventricular Internal Dimension Measurements for FSTL3 KO

Hearts Compared to WT Hearts.

Hearts were examined for changes in these measurements in response to exercise training, sex, and genotype. No significant differences were observed between groups with the exception of the WT and FSTL3 KO exercised females (*p<0.05). All measurements were within normal ranges. Figure produced in collaboration with Dr.

Zhaobin Xu. 109

Figure 33. Left Ventricular Internal Dimension Measurements for DKO Hearts

Compared to WT Hearts.

Hearts were examined for changes in these measurements in response to exercise training, sex, and genotype. No significant differences were observed between groups.

Figure produced in collaboration with Dr. Zhaobin Xu.

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Figure 34. Heart Weight to Body Weight (HW/BW) and Heart Weight to Tibia

Length (HW/TL) Ratios for FST288 Hearts Compared to WT Hearts.

Hearts were examined for changes in weight in response to exercise training, sex, and genotype. No significant differences were observed between groups with the exception of the male and female FST288 exercised hearts (* or ***p<0.05). FST288 = “KO.”

Figure produced in collaboration with Dr. Zhaobin Xu.

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Despite the differences in remodeling observed, male and female FST288 hearts show no changes in function in response to exercise training (Figure 36). Ejection fraction and fractional shortening were not significantly different between groups.

Ejection fraction and fractional shortening for all of the groups were within normal limits

(55-70% and greater than 25%, respectively). Although not significant, hearts from exercised female FST288 mice tended to have increased EF and FS. These results might be expected since the measurements were performed at rest. Experiments where load is placed on the heart may indicate more profound changes in function; thus, transverse aortic constriction (TAC) was next performed to determine the role of FST315 on hypertrophy in these hearts.

Mice were subjected to TAC, and hearts were analyzed by echocardiography, dissected after 4 weeks, extracted for protein, and analyed for changes in multiple signaling pathways. Interestingly, while the WT TAC hearts showed a significant increase in weight compared to the control WT hearts, the FST288 hearts subjected to

TAC were not significanty different from the control FST288 hearts or the WT control hearts (Figure 37). The hypertrophic markers, ANP and BNP, were significantly upregulated in the WT TAC hearts, but not in the FST288 TAC hearts.

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Figure 35. FST288 Hearts show Potential Differential Remodeling in Response to

Exercise Training.

Echocardiograph analysis of LVIDs (“Diameter; S”) and LVIDd (“Diameter; D”) indicated that FST288 heart left ventricular internal dimensions from exercised male mice were significantly elevated when compared to exercised female FST288 mice

(*p<0.05). Female mice responded to exercise training with a non-significant decrease in

LVID compared to the control. Figure produced in collaboration with Dr. Zhaobin Xu.

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Figure 36. FST288 Hearts show no Changes in Function in Response to Exercise

Training.

Despite the differences in remodeling observed, ejection fraction and fractional shortening were not significantly different between groups in either male or female hearts. Ejection fraction and fractional shortening for all of the groups were within normal limits (55-70% and greater than 25%, respectively). Figure produced in collaboration with Dr. Zhaobin Xu.

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Data from the echocardiogram analysis indicated that heart rates, cardiac output, and stroke volumes from all of the hearts were not-signficantly different from one another (Figures 38-39). However, WT hearts subjected to TAC showed significantly increased diastolic and systolic volumes, left ventricular mass, LVIDs, and LVIDd compared to the control WT hearts. On the other hand, the FST88 TAC hearts were not significantly different from the FST288 control hearts on any of these measures. Other measures of function, including ejection fraction and fractional shortening, were significantly decreased in the WT TAC hearts, but not in the FST288 TAC hearts.

Since the attenuation of the hypertrophic response observed in the FST288 hearts may be a result of altered phospho-AKT signaling, we examined the molecular pathways potentially influencing these observations. Cardiac myocyte lysates were subjected to

Western blotting (n=2 mice/group, experiment repeated three times). Because the AKT pathway is known to induce cardiac hypertrophy,252 we first probed with antibody against phospho- and total AKT. These hearts showed an alteration in the AKT signaling pathway (Figure 40), as pAKT signaling was reduced in the FST288 hearts subjected to

TAC versus the WT TAC hearts. We also assayed these samples by Western blot for changes in ERK signaling, but appreciable changes were not observed (Figure 40).

Finally, blots were probed for PI3K, and results showed that the FST288 groups were lower in PI3K than the WT groups, although inconsistencies within samples were observed (Figure 40).

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Figure 37. FST288 Hearts Display Blunted Hypertrophic Response after TAC.

WT and FST288 mouse hearts were subjected to TAC, analyzed by echocardiography, and weighed. WT heart weights were significantly increased (***p<0.05) after TAC, but

FST288 TAC hearts were not significantly different than FST288 or WT control.

Further, ANP and BNP were significantly increased only in the WT TAC hearts

(*p<0.05). Figure produced in collaboration with Dr. Zhaobin Xu.

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Figure 38. Structural Measurements in Response to TAC.

WT TAC hearts showed significantly (***p<0.05) increased volumes, left ventricular mass, and left ventricular internal dimensions when compared to WT control hearts.

FST288 TAC hearts (“FST TAC”) were not significantly different from the FST288 control hearts (“FST”) on any of these measures. However, FST288 TAC hearts were significantly lower on all of these measures when compared to the WT TAC hearts (* or

**p<0.05) except on the measure of LVIDd, indicating blunted hypertrophic remodeling in response to TAC in FST288. Figure produced in collaboration with Dr. Zhaobin Xu.

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Figure 39. Functional Measurements in Response to TAC.

Heart rates, stroke volumes, and cardiac output from all of the hearts tested were not significantly different from one another. WT TAC hearts displayed significantly decreased ejection fraction and fractional shortening (***p<0.05) compared to the WT control hearts. FST288 TAC (“FST TAC”) hearts were not significantly different from

FST288 control (“FST”) hearts on these measures, indicating blunted decrease in function in response to TAC in FST288. Figure produced in collaboration with Dr.

Zhaobin Xu. 118

Figure 40. Examination of Hypertrophic Signaling Pathways in Hearts Subjected to

TAC.

Results of Western blot analysis of cardiac myocyte extracts (n=2 mice/group; figure is representative of three independent experiments) showing that attenuation of the hypertrophic response in FST288 hearts may be a result of altered AKT signaling. The figure shows a decrease in pAKT in both FST288 control and FST288 TAC in comparison to the WT groups. PI3K signaling may also contribute to these changes in hypertrophy, as the FST288 groups showed decreased PI3K compared to the WT groups.

No appreciable changes in ERK signaling were observed. Figure produced in collaboration with Dr. Karthik Krishnamurthy. 119

We also tested for the involvement of TGF-β family member signaling using

Western blotting analysis. In this pathway, MSTN binds to its receptor (ActRIIB), and

Smad 2/3 is phosphorylated and activated. FST and FSTL3 can bind to MSTN and prevent MSTN from binding to its receptor. We did not observe significant differences in pSMAD 2/3, TGF-β1, MSTN, FSTL3, or activin (Figure 41) that would indicate that the TGF- pathway was the primary cause of the hypertrophic changes in response to

TAC.

Discussion

The primary focus of this study was to determine the role of circulating FST315 on the heart by using a transgenic mouse model expressing only the FST288 isoform.

Mice were subjected to TAC for 4 weeks, a protocol that is used as a model for pressure overload-induced hypertrophy and heart failure.253 After the 4 weeks, WT mouse hearts were significantly hypertrophied in comparison to sham WT mice, whereas the hypertrophic response was blunted entirely in the FST288 mice (Figure 37) subjected to

TAC. Hypertrophy also was not observed in sham FST288 hearts. Western blotting results indicated that the hypertrophic response was blunted in the FST288 mice due to altered pAKT signaling (Figure 40). Although WT mice subjected to TAC showed an increased expression of this pathway, FST288 TAC mice showed a decrease in pAKT signaling.

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Figure 41. Examination of TGF- Signaling Pathways in Hearts Subjected to TAC.

Results of Western blot analysis of cardiac myocyte extracts (n=2 mice/group; figure is representative of three independent experiments) showing no changes in FSTL3, TGF-

1, pSMAD2, or pSMAD3 signaling in response to TAC in any of the groups. Myostatin

(MSTN) and activin appear to be co-regulated, but not in response to TAC.

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Signaling pathways controlling hypertrophy are complex and crosstalk can occur, but AKT is recognized as a major contributor to hypertrophy in the heart.254 Postnatal cardiac growth occurs as a result of myocyte hypertrophy versus hyperplasia, and the

AKT signaling pathway contributes to this cardiac hypertrophy.255 In response to growth factor stimulation, AKT becomes activated by phosphorylation, then translocates to various sites within the cell and phosphorylates downstream substrates. AKT phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3), an inhibitor of cell growth pathways, resulting in hypertrophy. In contrast, ERK1/2, another recognized mediator of cardiac hypertrophy, is not necessary for development of hypertrophy.254

Indeed, we did not observe significant changes in ERK signaling in response to TAC or loss of FST315 (Figure 40). These results are unsurprising given that signaling through

PI3K/AKT also has been shown to inhibit ERK signaling.256

FST has been shown to mediate skeletal muscle hypertrophy through the

AKT/mTOR pathway in duck myoblasts and in mice.257, 258 In the mouse study, FST288 was overexpressed by administration via adeno-viral vector directly to skeletal muscles to increase muscle hypertrophy. The effect was attenuated with a constitutively active

Smad3, a downstream inhibitor of the AKT pathway. FST288 increased muscle hypertrophy through its influence on the AKT pathway regardless of the presence of

MSTN. While it is known that FST can mediate muscle hypertrophy by binding MSTN and activin and preventing their activity, which includes modulation of mTOR signaling, we observed no changes in the protein levels of MSTN, activin, or other members of the

TGF- family or signaling pathways in our heart lysates (Figure 41) that would indicate

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that this pathway is responsible for the observed effects. These results are consistent with the findings of Winbanks et al.,258 suggesting that FST may be contributing to muscle hypertrophy through a mechanism other than MSTN inhibition.

FST family members bind to and modify TGF- family members. FSTL3, for example, has been shown to bind to activin A and affect the heart’s response to pressure overload and ischemia.197 However, our studies did not show changes in the downstream mediators of TGF- signaling, including pSmad2 and pSmad3, in spite of the loss of

FST315.

We also examined the potential role of PI3K in modulating the growth of the hearts in this study. We found reduced levels of PI3K in the FST288 mice overall, although these results were not consistent. Future studies, including repeat analysis of

PI3K Western blots, as well as pPI3K Westerns, will enable a more clear picture of the role of PI3K in these models. In the case of signaling involving PI3K, a growth factor binds to its receptor tyrosine kinase, PI3K is phosphorylated, and AKT is phosphorylated and activated, leading to activation of mTORC1, which phosphorylates effectors to result increased protein production and cell survival.259

Hearts from the WT mice subjected to TAC showed not only hypertrophy and altered signaling, but also increased biomarkers and phenotypic changes indicative of heart failure including increased ANP, BNP, diastolic and systolic diameters and volumes, left ventricular mass, LVIDs, and LVIDd (Figure 38). Fractional shortening and ejection fraction were significantly decreased in these mice. These changes were expected in the WT mice in response to TAC, but the loss of FST315 appeared to be

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protective. FST288 mice showed only minimal signs of these pathological changes associated with heart failure. This result is supported by the apparent increase in exercise-linked, physiologic hypertrophy in FST288 mice. The hypertrophic signaling in the heart of the FST288 must remain intact; however, they do not progress into pathologic decompensation during heart failure. To our knowledge, this is the first study to characterize the role of FST315 in the heart and in a model of induced pressure overload. Our results are not surprising considering that follistatin has been determined to be important for skeletal muscular growth,258, 65 thus a partial knockout of this system might be expected to decrease hypertrophy by reduction in signaling through the AKT pathway. Examination of histologic staining in these hearts to compare degree of fibrosis and myopathy to WT hearts is warranted.

We also examined the response of the heart to alterations in FSTL3, with and without exercise. Although the literature shows that systemic FSTL3 KO mice show elevated heart weight to body weight ratios compared to WT mice,31 our data did not show this increase in FSTL3 KO mice (Figure 30). Further, it has been reported that

FSTL3KO mouse hearts also showed significantly increased left ventricular end systolic pressure and systolic arterial pressure, indicating that these animals are hypertensive. We did not observe these pathological changes in the resting state, and in fact, observed that our FSTL3 KO mice had normal ejection fraction, fractional shortening, LVIDs, and

LVIDd (Figures 31-32), although pressures were not measured. We did see higher

LVIDd and LVIDs in female FSTL3 KO exercised mice compared to the female WT exercised mice. While Schneyer et al., 2007 did not explore the observations of

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pathology in their FSTL3 KO mice further, we also analyzed myocyte extracts for altered pAKT and pGSK signaling. We observed a decrease in the signaling of these hypertrophic pathways in our FSTL3 KO mice (data not shown). The discrepancies between these two studies may simply reflect the age of the mice, as our mice were approximately 4-6 months old, whereas the mice in the Schneyer study were 9 months old. Future studies should determine the time course of development of these pathological changes in the FSTL3 KO mice. Taken together, these results, along with the normal function of the DKO hearts observed in our study (Figure 33), suggest that the

FSTL3 and FST315 mediators are serving distinct functions in the heart. If FST315 and

FSTL3 were serving the same functions, we would have expected the DKO hearts to display any pathologies in an additive manner.

FST288 hearts also were examined for potential differential effects to exercise training. HW/BW and HW/TL ratios between FST288 and WT mice were not significantly different (Figure 34). Interestingly, the male FST288 exercised mice had larger hearts that the female FST288 exercised mice, although these hearts were not significantly larger than the exercised WT male hearts. Due to these unclear results concerning heart weight and potential trends towards differential sex effects, we decided to further study the FST288 hearts. Although the EF and FS in all male or female

FST288 mice were normal (Figure 36), whether they had been exercised trained or not, differences in LVIDs and LVIDd in males and females in response to exercise training were observed (Figure 35). Exercised male FST288 mice had increased LVIDs and

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LVIDd compared to exercised female mice. These results show potential differential sex effects on the heart due to the loss of FST315.

The role of estrogens on the heart have generally been regarded as being protective, and although evidence to the contrary has been observed, it appears that estrogens work through multiple signaling pathways to inhibit pathological growth.260

However, female mice have been shown to have increased physiological hypertrophy in response to exercise training.261 The fact that our female FST288 mice have an LVID response to exercise training similar to that of control mice is interesting, and may simply be a reflection of the protective effects of the female sex on heart function. As this study is the first to closely examine the heart of the FST288 mouse model, further study on sex differences in these mice is warranted.

The expression level of FST itself may be playing a role in sex differences in these mice. FST mRNA is expressed ubiquitously in the rat,262 including ovary, testis, cerebral cortex, pituitary, adrenal, thymus, pancreas, gut, kidney, heart, uterus, skeletal muscle, lung, etc.,46 but there is a difference in the amount of FST in the ovary and kindey versus the testis,46 indicating that a sex difference does exist. In the FST288 mouse model in particular, there are differences in the KO response of FST315 in males versus females, as FST expression in the ovaries was reduced by half, but there were no changes in expression in the male testes.48 However, due to its affinity for heparin sulfate, the cellular localization of FST is not necessarily indicative of production by that cell type. Nor does detection of FST mRNA guarantee translation into protein.262

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Nevertheless, this difference in expression level could account for the differential results obtained for these mice.

The response of these transgenic mice to execise further complicates the issue. A few studies on the follistatins and exercise have been described, and these studies show that the response of FST to exercise are complex. Plasma FST, thought to be originating from the liver, has been increased in response to acute cycling exercise in humans,43 but no changes were observed in the muscle. Another study showed that FST liver mRNA expression was determined to be lower in swim trained rats than in control rats.263 No change in FST mRNA was found in the muscle of young women after a single bout of resistance exercise.82 Although this should be studied further, a simple up or downregulation of FST288 from basal level in specific tissues in response to exercise could also account for a differential response between males and females in these mice.

Conclusions

Mechanisms of heart remodeling and failure are important to determine, as this is a significant health problem in the U.S. The presence of secreted factors in the heart that contribute to pathological hypertrophy are increasingly identified and investigated. In this particular study, we examined a potential novel mechanism contributing to changes in the heart in response to exercise and pressure induced overload involving the follistatin and related proteins. Using multiple transgenic mouse models, we show that loss of a particular FST isoform, FST315, abolishes cardiac hypertrophy observed after TAC- induced pressure overload. We found that this effect is a result of reduced AKT pathway

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signaling, and that the FST isoform FST315 is one contributor to pathological cardiac hypertrophy.

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Chapter 5: Translational Potential of Walking Exercise in Humans

Introduction

Musculoskeletal (MSK) disorders are a significant health problem in the United

States. Approximately 48% of the U.S. population has experienced a MSK disorder at a collective cost of over $950 billion in direct and indirect expenditures annually.328 These common and costly disorders, which include functional impairments of the muscles, bones, joints, tendons, and/or ligaments,329 markedly decrease quality of life.39 Adverse consequences of MSK disorders include work absences, chronic pain, decreased quality of life, and substantial economic burden through direct medical expenditures as well as decreased productivity.264, 265 The prevalence of MSK disorders is expected to increase due to an upsurge in the aging population in the U.S. 266 demonstrating that new treatment options for these disorders are needed critically.

Exercise is beneficial for strengthening muscle and bone and frequently is prescribed as a treatment option for MSK disorders.267, 6, 8, 7 However, as discussed in previous chapters, the mechanisms by which exercise exerts its effects are not well known or studied. Further, the most beneficial type and amount of exercise for improving the MSK health of an individual patient is unknown. We have attempted to define a molecular mechanism of exercise during our previous studies of the mechanosensitive protein, FSTL3, in the mouse.30 The molecular pathways affected by this protein also have the potential to regulate muscle formation and strengthening, 129

indicating that exercise can regulate muscle and bone through a common mechanism.

Therefore, the goal of this study was to determine a molecular mechanism that collectively regulates bone and muscle formation and strengthening in response to exercise in humans. Understanding such mechanisms is vital for development of new treatment strategies for MSK disorders and to provide new tools for clinical assessment of MSK health. Our central hypothesis was that exercise regulates bone and muscle growth and strength via FSTL3-mediated mechanisms. The long-term impact of these studies would enable effective exercise prescriptions to treat MSK disorders and the development of FSTL3 as a biomarker for assessment of MSK health. FSTL3 could also be developed as a potential therapeutic for MSK disorders.

Molecular indicators of the efficacy of exercise in producing gains in muscular strength and bone mass are lacking. Relatedly, individualized exercise prescriptions for optimal health and rehabilitation are lacking despite the known heterogeneity of responses to exercise training.11 Studies are needed to determine the appropriate intensity, frequency, duration, and mode of exercise to increase health,11 and importantly, knowledge of physiological factors responsible for variability in exercise responsiveness are needed.12 A relevant gap in the literature is the absence of objective clinical outcome measures that could inform tailored exercise interventions.12 A consistently cited critical barrier to progress in the field of clinical therapeutic exercise for MSK disorders is the lack of a personalized therapeutic exercise prescription for patients containing appropriate type, duration, frequency, and mode of exercise to treat the specific MSK condition.5, 4, 268, 269 A lack of valid and reliable outcome measures further complicates

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treatment options,5 and only limited information exists on the molecular mechanisms of exercise therapy that could inform exercise prescriptions.4

Although exercise has been prescribed for MSK health for years, scant knowledge linking the molecular effects of exercise to training dose and health outcomes is available.270 The current clinical paradigm for treatment of MSK disorders uses outcome measures such as range of motion, swelling and pain, restored strength, and quality of life to determine patient health.271, 272, 273, 274, 275 The use of objective criteria after injury, such as strength symmetry and performance on functional tests, are markers by which clearance for activity and progression of rehabilitation is measured.276 When prescribing training to healthy adults for optimal MSK health, personal trainers use exercise guidelines implemented by national organizations such as the American College of

Sports Medicine. These guidelines are based on training variables such as frequency, intensity, duration, and mode of exercise as well as training principles of overload, progression, etc.330 However, training response variability exists.270 In all of these instances, objective, valid, and reliable measurements are necessary to assess the effectiveness of exercise as well as to inform the most appropriate interventions. The lack of specific, measureable, appropriate indicators of exercise effectiveness leads to an inefficient, potentially ineffective “trial and error” approach to exercise prescription. A need exists for a shift in the clinical treatment paradigm for MSK health from reactive i.e., therapy based on outcome measures of current practices, to proactive, i.e., prediction of phenotypes based on molecular markers and earlier implementation of appropriate and personalized therapies.

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We intended for this study to create an early framework for the exploitation of one such molecular exercise mechanism to improve exercise prescriptions and MSK disorder treatment options. We observed that the FSTL3 mediator had potential as a clinical serum biomarker for quantitation of muscle and bone health (growth and strength) in both healthy and diseased individuals because our previous studies showed

FSLT3 to be present and tightly regulated in the serum,30 thus indicating great potential for its role as a biomarker. We also considered that FSTL3 in serum could be used clinically to gauge the success of exercise in promoting muscle and bone growth and strength, as a suitable exercise prescription would be expected to increase circulating levels of this biomarker. In this manner, personalized exercise prescriptions for MSK diseases could be developed.

FSTL3 is related in structure to follistatin (FST), and both of these molecules are upregulated in response to exercise and play roles in regulating transforming growth factor beta (TGF-β) family members. FSTL3 binds and antagonizes the TGF-β molecules activin and myostatin (MSTN or GDF8).31 Since MSTN inhibits myogenesis, the inhibition of MSTN potentially could promote muscle hypertrophy.277 FSTL3 is a known inhibitor of MSTN 32, 33 that is upregulated in response to exercise.177 FSTL3 is structurally similar to FST but lacks a follistatin domain, lending unique properties to these two related proteins.39 FSTL3 is expressed in most tissues, especially in the placenta and testis.61 MSTN, found primarily in skeletal muscle and circulating in serum, is an inhibitor of myogenesis. MSTN ablation results in both hyperplasia and hypertrophy of muscle fibers.64 MSTN signals by binding to and activating its receptor,

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activin type IIb (ActRIIB), which then activates Smad proteins. Smads regulate myogenic gene expression and can inhibit MyoD, a transcription factor that increases myoblast proliferation. In addition, myostatin inhibits the Akt/insulin-like growth factor

(IGF-1) pathway, resulting in inhibited protein synthesis (inhibits mammalian target of rapamycin, mTOR, pathways) and increased ubiquitin ligase pathways (through FoxO1 transcription factors). The end result is increased muscle atrophy and decreased muscle hypertrophy.70 MSTN expression has been shown to decrease in response to exercise.77,

78 FSTL3 neutralizes MSTN by preventing binding of MSTN to ActRIIB, thus preventing downstream signaling responsible for inhibition of muscle differentiation and hypertrophy.86, 34, 65

Due to our success in modulating short-term (15 day) bone growth in mice through walking exercise-mediated FSTL3 pathways,30 we aimed in this study to determine longer-term (6 weeks) impacts of walking exercise on FSTL3 expression and changes in bone mass and muscular strength in humans. We also compared walking exercise to another exercise mode with the goal of increasing FSTL3-associated musculoskeletal growth and strength through the more beneficial mode of exercise.

Walking as a therapy for low bone density and muscle sarcopenia

Exercise is beneficial for maintenance of overall health. Studies have shown that moderate walking exercise (3-5 mph) imparts a multitude of health benefits including improvement in blood pressure, weight maintenance, decreased depressive symptoms, and increased cardiovascular health. Walking also decreases chronic musculoskeletal pain, increases bone mineral density, and can decrease the sarcopenia observed with

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aging.278, 279, 280, 281, 282 Exercise is beneficial for strengthening muscle and bone and can be prescribed as a treatment for musculoskeletal disorders.7, 6, 8 Walking exercise is an attractive option because it is low impact and has low potential for injury. Studies are inconclusive on the effectiveness of walking exercise for increasing bone mineral density

(BMD) and muscle strength. A recent systematic analysis on the effects of exercise in general on vertebral fractures was mixed.283 When specifically considering the effects of walking, a couple of studies indicate no evidence that walking increases BMD, other than at the femoral neck.284, 285 In fact, walking alone may not be effective for increasing

BMD without a concurrent incorporation of other forms of physical activity,286 although one study found higher overall BMD with a one mile per day walking program.287 A meta-analysis on the effects of walking showed a modest increase in BMD in osteoporosis, but showed that an additional stimulus (strength, high impact, whole body vibration training) would be beneficial for observing the greatest BMD gains.288 In a study comparing females in various sports, high intensity exercise was determined to be the most beneficial for bone.289

Similar to observations in bone, data on muscular strength improvements with walking also are inconclusive.282 Walking has been shown to be an insufficient stimulus to increase BMD, aerobic fitness, and muscle strength.290 However, a different study showed that muscle strength improved after 6 months of walking training, indicating that a long term intervention may be necessary.291 In spite of the contradictory results observed in these studies, walking remains a “weight-bearing” exercise of choice for treatment of osteoporosis/osteopenia due to its ease, safety, and accessibility to the

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general population.292, 293, 331 Therefore in this study, we aimed to further investigate the effects of a six week walking exercise program on BMD, muscle strength, and serum biomarkers of musculoskeletal health. To our knowledge, despite the existence of many meta-analyses on the topic of walking, no single study exists that connects a controlled walking program, BMD, muscle strength measures, and blood biomarkers.

Furthermore, we aimed to compare the results of this walking protocol to a protocol that was aimed to decrease the propensity for ACL tear using a neuromuscular, jumping, cutting, balance, trunk and leg strengthening, core strengthening, and plyometric training program (“P4ACL”) during 12, 45-minute sessions over approximately eight weeks. The question of the best type and amount of exercise to produce the strongest but lightest skeleton is a compelling one that has been hotly debated for some time.294 The P4ACL protocol was chosen because studies suggest that greater gains in bone density and muscle strength are obtained with higher impact activities.295, 296, 297, 298, 299

In fact, different types and intensities of exercise differ in their effects on bone and muscle. High-intensity300, 301 long duration exercise regimens302, 284, 303 provide greater bone-building benefits than low-intensity, short-duration regimens; however, the molecular pathways controlling these adaptations to specific exercise stimuli are not defined. FSTL3 may be differentially regulated as exercise stimuli change. FSTL3 serum levels are regulated in response to exercise, but no long-term studies on its regulation have been performed.177 Although high-intensity, high-impact exercise has been shown to be beneficial for increasing BMD,295, 296, 297, 298, 299 the relative

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contributions of aerobic and resistance training to these BMD effects are not well quantified. Resistance training has been shown to influence both BMD and muscle strength,304 and MSTN inhibition appears to be regulated by exercise type, intensity, and training status.70 In this study, we tested the relative changes in FSTL3 and MSTN in response to two types of exercise training.

Methods

Recruitment

Subjects were males and females between the ages of 14-49 in the Central Ohio metro area. We attempted to recruit 60 subjects (20 per group) to obtain sufficient power as well as to account for potential dropout. One group of participants was otherwise healthy, but sedentary, and were interested in starting a walking program to improve their health in general. These participants were recruited using recruitment flyers posted in university common areas (i.e. bulletin boards in the student union) and local businesses

(i.e. gym facilities, running stores, etc.), via ResearchMatch.org, a national, electronic web-based recruitment tool maintained at Vanderbilt University, and via StudySearch, another web-based recruitment tool local to OSU. Another group of participants consisted of competitive walkers from the Central Ohio area, specifically including the

New Albany Walking Club. Finally, the third group consisted of P4 ACL participants.

These subjects were enrolled in a paid program to decrease their susceptibility to knee/anterior cruciate ligament (ACL) injury through core, neuromuscular, and plyometric training. Participants were compensated $25 for each study visit, for a total of

$100.

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Exercise protocols

Exercise protocols consisted of the following: 1. Beginner (previously sedentary) walking exercise group (Low intensity/low impact, LILI). This group walked for 45 min/day for 6 weeks from 2.5-3.5 mph. 2. Advanced walking EX group (High intensity/low impact, HILI). This group walked for 45 min/day for 6 weeks at a speed >

3.5 mph. 3. High-intensity plyometric (explosive power) EX group (High intensity/high impact, HIHI): These subjects were recruited from the OSU Sports Medicine P4 ACL

Injury Prevention Program. Subjects were trained using a variety of jumping, cutting, core strengthening, plyometric, balance, and trunk and leg strengthening EX during 12,

45-minute sessions over approximately six to eight weeks.

Serum analyses

Blood was collected from each subject before the start, after the first week, at the mid-point, and at the end of the exercise intervention. The serum was separated and stored at -80C until analyzed. Serum was tested for FSTL3, FST, and MSTN by commercial enzyme-linked immunosorbent assay (ELISA) (Human FLRG Quantikine

ELISA Kit, R&D Systems, Minneapolis, MN; Human Follistatin ELISA Kit, Abcam,

Cambridge, MA; and Human Myostatin ELISA Kit, Mybiosource, San Diego, CA).

Measurement of bone mineral density

Bone mineral density was assessed both before and after the exercise interventions using a General Electric (GE) Lunar iDXA dual-energy X-ray absorptiometry (DXA) scanner (GE Healthcare, Little Chalfont, Buckinghamshire,

United Kingdom) by a certified technician at OSU to compare bone densities of the

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spine, wrist, and femur among the intervention groups. A series of 4 scans were conducted in order to accurately measure the amount and distribution of body fat mass, lean mass, and bone. These tests administer a small amount of radiation, similar to the amount of radiation received during an airplane flight from New York to Los Angeles.

The GE Lunar iDXA used in this study is a particularly low-dose DXA device, administering 95% less radiation than typically-administered radiation levels estimated by the Duke Radiation Safety Committee. DXA scanning was only conducted on males, or females who provided recent (results dated <4 weeks prior to DXA scanning) urinalysis testing results from a primary care physician or licensed clinician verifying that they are not currently pregnant, in order to prevent undue exposure to females who may be unknowingly pregnant.

Assessment of muscular strength

Muscular strength of the quadriceps and hamstrings was assessed using Biodex isokinetic dynamometry (Biodex Medical Systems, Inc., Shirley, NY) both before and after the exercise interventions. Subjects were seated on the dynamometer with their knee flexed to 90°. After undergoing a short warm-up, subjects pushed and pulled against a padded buttress and a strap positioned just above the ankle. Immobilization on the Biodex seat consisted of a thoracic strap, waist strap, bilateral thigh straps, and a shin strap. Testing was conducted at 60° and 300° per second to mimic various sports movements. The range of motion defined by the manufacturer is 100° with 0° representing full extension. Peak and average flexion and extension torques were

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assessed. Peak torques of the muscle groups were compared among the intervention groups.

Muscular strength of the hip, including isometric hip abduction and external rotation strength were determined using a custom force sensing strap. A non-stretchable cloth band was secured around the subject’s thighs, superior to the femoral condyles and patella. Hip abduction and external rotation strength tests were performed in standing, sitting, and lying positions. The subjects maximally abducted their thighs and pushed out against the strap, and two maximum efforts were recorded for each test. A strain gauge measured the induced voltage and displayed force output on a display. Maximum force during the two trials was recorded and averaged.

Statistical analyses

The relationship between serum FSTL3 concentration and BMD was examined for significant correlations using GraphPad Prism v 4.0 (GraphPad Software, San Diego,

CA). Results were considered statistically significant when p < 0.05. One-way Analysis of Variance (ANOVA) was used to examine differences between groups for the DXA and strength experiments. The Kruskal-Wallis nonparametric test was used to compare group means for the ELISAs. Within group effects were examined using pairwise t-tests.

Results

Study population

Table 9 and Figure 42 show the characteristics of the recruited subjects. Most of the subjects were female (n=19, male n =1) and between the ages of 14-49. The SED group contained the most subjects (n=13), the CON group contained 4 subjects, and the

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P4ACL group contained 3 subjects. The P4ACL group contained the youngest participants, as this group tended to be high school or college athletes or recreational athletes. The SED group ranged in age from 20-49.

Bone mineral density

Table 10 and Figure 43 show the total body bone mineral density (g/cm2) as well as the BMD for each body site (Figure 44) for each participant in the three groups.

Neither the exercise groups nor the control walking group resulted in any significant measureable change in bone mineral density in any of the measured sites or the total body measurement after the 6-8 week interventions. Similarly, there was no significant differences between groups. Further, FSTL3 serum concentrations were analyzed for correlation to total BMD measurements, and resulted in a correlation coefficient of

0.1407 (Figure 45). Similarly, BMD of the spine and femur also did not correlate to serum FSTL3 concentration (coefficients of 0.211145 and 0.178529, respectively).

However, the BMD of the forearm weakly correlated to serum FSTL3 concentration (0.3,

Figure 45).

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Table 9. Subject Characteristics

Group Age Height (in) Weight (lb) Sex NA 43 63.25 190.2 F NA 49 66.1 165.8 F NA 47 65 143.6 F NA 42 64.5 146.6 F P4 ACL 14 66 145.4 F P4 ACL 14 67.9 127.2 F P4 ACL 14 64.4 160.8 F SED 43 61.02 166 F SED 49 62.5 157.4 F SED 33 67.7 161.6 F SED 34 75.5 186 M SED 20 63 111.4 F SED 41 69.7 264.8 F SED 21 62 162.4 F SED 48 62.5 171 F SED 29 66.6 222.2 F SED 23 67.75 112 F SED 48 64 203.6 F SED 26 66 138.6 F

SED 38 59.5 126 F

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Figure 42. Average Subject Characteristics.

The P4 ACL group was the youngest, with an average age of 14, whereas the NA walking control group was the oldest (average age 45). The P4 ACL groups also was the tallest (average height 66.1 in) with a range in all groups from 62.75-66.1 in. The sedentary group was the lightest, with weights ranging from 132.3-161.55 lb for all of the groups.

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Table 10. Average Bone Mineral Density (g/cm2)

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Figure 43. Total Bone Mineral Density Results.

The individual percent differences from pretest to posttest are presented for each subject in the top chart. The bottom chart shows the average percent differences for each group.

None of the groups displayed a significant within group change in BMD after the intervention. There were no significant differences in percent BMD between the groups.

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Figure 44. Bone Mineral Density Site Results.

Average percent differences for each group for the spine, femur, and radius are shown.

None of the groups displayed a significant within group change in BMD after the intervention. There were no significant differences in percent BMD between the groups. 145

Figure 45. Correlation between FSTL3 Serum Concentration and BMD.

FSTL3 serum concentration was tested for correlation to total BMD as well as BMD at each site. Pictured are the scatterplots showing the relationship between total BMD and

FSTL3 concentration and radius BMD and FSTL3 concentration. Serum FSTL3 did not correlate to total BMD (correlation coefficient = 0.14), but weakly correlated to radius

BMD (0.30). BMD of the spine and femur also did not correlate to FSTL3 concentration

(not pictured).

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Muscular strength tests

Results of the hip strength testing of the outer hip (abductor) muscles indicated no significant differences between any of the groups in response to the exercise interventions

(Figure 46) except the following: the P4ACL group and sedentary group and the CON walking and sedentary group were significantly different from each other when standing external rotation was tested (p=0.04 and 0.02, respectively). The following within group differences were also significant: side lying abduction left – sedentary was higher at post test (p=0.04), side lying external rotation right – sedentary was higher at post test

(p=0.03), standing abduction left – both control walking and sedentary groups were higher at post test (p=0.002 and 0.02, respectively), standing abduction right – sedentary was higher at post test (p=0.03), and standing external rotation – sedentary was higher at post test (p=0.0005).

Results of the Biodex testing (quadriceps and hamstrings) showed significant differences within primarily the SED group in response to the exercise interventions

(Figures 47-48). For example, at 60/sec extension, the sedentary group significantly improved torques on the right side (p=0.03), and on the left and right side during 60/sec flexion (p=0.02 and 0.0005, respectively). Similarly, the sedentary group improved on the left and right sides during 300/sec extension (p=0.03 and 0.03, respectively), and

P4ACL and CON groups improved on the right side (p=0.007 and 0.02, respectively).

There were no significant differences between groups other than the P4 group in which

60/sec extension on both sides was significantly different than in those in the sedentary

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group (p=0.004 left and 0.002 right). No significant differences between groups were noted at 300/sec torques.

Serum analysis

Table 11 and Figure 49 show the results of the FSTL3 ELISA assays. The standard curves for these assays performed acceptably (r2 values of 0.9982 and 0.9985), with a curve range from 62.5-4,000 pg/mL. All experimental values obtained for serum

FSTL3 concentration were within the range of the kit (3142-8554 pg/mL, mean of 6481 pg/mL, n = 38, SD = 1192 pg/mL). This assay had been extensively tested by the manufacturer for cross reactivity to similar molecules, such as FST, and no significant cross reactivity was observed (< 0.5% cross-reactivity observed with available related molecules). Average serum FSTL3 concentrations between the CON walking group and the P4ACL or sedentary groups was not significant when analyzed by ANOVA (p>0.05).

However, the average serum concentration in the P4ACL group was significantly less than the sedentary group (p=0.01). It should be noted that the P4ACL group contained a small number of participants, therefore the significant difference between this group and the sedentary group may not be a true effect. Pairwise, non-parametric Mann-Whitney tests were also peformed to determine any significant changes occurring within groups at each point in time. No significant differences between points were observed.

We next wanted to determine levels of MSTN in the serum (Table 12, Figure 50), as we expected FSTL3 to bind to MSTN in the serum. The standard curve for this assay performed acceptably (r2 value of 0.9602), with a curve range from 0.625-20 ng/mL.

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Figure 46. Hip Strength Testing Results.

Within group differences (*p<0.05) for the hip abductor muscles indicated that the sedentary group significantly improved on multiple measures. No between group differences were significant except in standing external rotation (lines represent differences significant at p<0.05).

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Figure 47. Biodex Testing Results at 60 Degrees per Second.

Significant within group differences for the sedentary group were noted during extension on the right side and flexion on both left and right sides (*p<0.05). Strength in this sedentary group improved from pre to post for all of these measures. Significant between group differences were observed for extension on both right and left sides (p<0.05 represented by the bar) between the P4 ACL and sedentary groups.

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Figure 48. Biodex Testing Results at 300 Degrees per Second.

Significant within group differences for the sedentary group were noted during extension on both the right and left sides, and for the NA walking control and P4 ACL groups on the right side (*p<0.05). No within group differences were noted for the flexion exercise, nor were any groups significantly different from one another.

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Table 11. Serum FSTL3 Concentration (pg/mL)

Subject Group Time Point Concentration (pg/mL) 1 NA 1 6556.7753 1 NA 2 6877.049 1 NA 3 6889.1348 1 NA 4 6004.8571 3 SED 1 6205.27995 3 SED 2 7018.05 3 SED 3 6013.92145 3 SED 4 7996.9998 4 SED 1 8869.1917 4 SED 2 7456.16025 4 SED 3 7675.71895 4 SED 4 8143.03655 5 SED 1 8525.75355 5 SED 2 7212.42995 5 SED 3 7887.22045 5 SED 4 7840.89155 6 SED 1 5316.97365 6 SED 2 5368.3383 6 SED 3 5770.19115 6 SED 4 5785.2984 7 SED 1 6084.42195 7 SED 2 6136.79375 7 SED 3 6277.79475 7 SED 4 6167.00825 9 SED 1 8948.75655 9 SED 2 8610.35415 9 SED 3 9166.30095 9 SED 4 9451.3244 10 SED 1 5562.71825 10 SED 2 5992.7713 10 SED 3 5958.5282 10 SED 4 5223.3087 11 P4 1 6416.78145 11 P4 2 6785.39835 11 P4 3 6749.14095 11 P4 4 7139.91515 12 P4 1 5422.3212 12 P4 2 6171.4956 12 P4 3 5035.8108 12 P4 4 6190.374 13 P4 1 5389.5324 13 P4 2 5222.6076 13 P4 3 5277.2556 13 P4 4 5311.038 14 NA 1 7655.934 14 NA 2 7694.6844 14 NA 3 7593.3372 14 NA 4 8743.926 15 NA 1 6809.3868 15 NA 2 7275.3852 15 NA 3 7644.0108 15 NA 4 6583.8396 17 NA 1 4514.1708 17 NA 2 4957.3164 17 NA 3 4909.6236 17 NA 4 4945.3932 18 SED 1 10001.2283 18 SED 2 9087.74325 18 SED 3 8155.12235 18 SED 4 6353.331 19 SED 1 7939.11 19 SED 2 8967.486 19 SED 3 8359.4028 19 SED 4 10802.6652 20 SED 1 5918.1276 20 SED 2 5633.958 20 SED 3 6105.918 20 SED 4 6472.5564 21 SED 1 8183.5356 21 SED 2 7700.646 21 SED 3 6955.446 21 SED 4 8962.518 23 SED 1 5868.4476 23 SED 2 6886.8876 23 SED 3 6335.4396 23 SED 4 6046.302 25 SED 1 4492.3116 25 SED 2 5040.7788 25 SED 3 4857.9564 25 SED 4 4938.438 152

Figure 49. Comparison of Serum FSTL3 Concentrations between and within

Groups.

Average serum FSTL3 concentration was not significantly different between the CON

(“NA”) walking group and the experimental groups; however, average serum FSTL3 concentration in the P4 ACL group was significantly less than in the sedentary group

(p<0.05). No significant within group differences were observed.

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Similar to the FSTL3 results, the average MSTN serum concentration in the P4ACL group was significantly less than in the sedentary group (p<0.05). Additionally, there was no correlation between serum FSTL3 and MSTN levels (correlation coefficient =

-0.11). Pairwise, non-parametric Mann-Whitney tests also were peformed to determine any significant changes occurring within groups at each point in time. No significant within group effects were noted. It should be noted that the dataset was small for the

P4ACL group (n=1) due to low recruitment in that group as well as values obtained outside of the assay standard curve that must be reassessed.

Finally, we determined serum concentrations of another MSTN binding partner,

FST (Table 11, Figure 51). In contrast to FSTL3 and MSTN concentrations, the serum

FST concentrations appeared to differ more widely within individuals. Similar to FSTL3 and MSTN results, the concentrations of FST over time did not appear to change in a predictable way with the training interventions. Average serum FST concentrations between the CON walking group and the P4ACL group was not significantly different

(p>0.05). However, the average serum FST concentraton in the sedentary group was significantly less than in the P4ACL group (p<0.05). There was no correlation between serum levels of FST and MSTN (correlation coefficient = -0.14) or between serum FST and FSTL3 (correlation coefficient = -0.01). The standard curve for this assay performed acceptably (r2 value of 0.994), with a curve range from 0.512-125 ng/mL. This assay had been extensively tested by the manufacturer for cross reactivity, and no significant cross reactivity was observed. No within group differences were observed.

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Table 12. Serum MSTN Concentration (ng/mL)

Subject Group Time Point Concentration (ng/mL) 1 NA 1 2.01898726 1 NA 2 2.09360923 1 NA 3 2.24823133 1 NA 4 0.36856441 3 SED 1 4.56796633 3 SED 2 9.349909111 3 SED 3 7.424417504 3 SED 4 9.485674048 4 SED 1 7.118407646 4 SED 2 8.845639345 4 SED 3 4.07123906 4 SED 4 8.476057016 5 SED 1 -1.218205986 5 SED 2 0.02091844 5 SED 3 -1.228980981 5 SED 4 -0.275393923 6 SED 1 15.61094212 6 SED 2 13.15513981 6 SED 3 18.00489559 6 SED 4 11.30505277 7 SED 1 20.70069829 7 SED 2 20.12254609 7 SED 3 33.81130783 7 SED 4 20.77599253 9 SED 1 9.791683906 9 SED 2 8.492219509 9 SED 3 7.708877372 9 SED 4 9.22168667 10 SED 1 -0.748416204 10 SED 2 -1.171873507 10 SED 3 -0.183806466 10 SED 4 1.322537836 11 P4 1 -0.90337043 11 P4 2 -0.26471393 11 P4 3 -0.66807593 11 P4 4 -1.62471614 12 P4 1 -0.16723478 12 P4 2 -0.6889163 12 P4 3 -1.233455 12 P4 4 -1.7107667 13 P4 1 4.6757983 13 P4 2 4.53999976 13 P4 3 4.77798334 13 P4 4 2.15814715 14 NA 1 8.438344534 14 NA 2 9.905898853 14 NA 3 5.053918604 14 NA 4 3.836344169 15 NA 1 1.309607842 15 NA 2 2.813797144 15 NA 3 -0.969303601 15 NA 4 1.330080332 17 NA 1 16.69867498 17 NA 2 16.45867459 17 NA 3 13.25329123 17 NA 4 9.70303336 18 SED 1 14.77799959 18 SED 2 11.16925423 18 SED 3 7.9363078 18 SED 4 12.85597966 19 SED 1 2.87008108 19 SED 2 7.3783237 19 SED 3 4.57025191 19 SED 4 2.07814702 20 SED 1 -2.28354074 20 SED 2 -2.22774233 20 SED 3 -2.20622969 20 SED 4 -2.26001129 21 SED 1 5.258643509 21 SED 2 9.081611735 21 SED 3 4.371861421 21 SED 4 3.765229202 23 SED 1 2.231947414 23 SED 2 1.435675283 23 SED 3 2.378487346 23 SED 4 0.672805637 25 SED 1 17.10036301 25 SED 2 9.608508991 25 SED 3 13.06728239 25 SED 4 13.81829954

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Figure 50. Comparison of Serum MSTN Concentrations between and within

Groups.

Average serum MSTN concentration was not significantly different between the CON

(“NA”) walking group and the experimental groups; however, average serum MSTN concentration in the P4 ACL group was significantly less than in the sedentary group

(p<0.05). No significant within group differences were observed.

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Table 13. Serum FST Concentration (ng/mL)

Subject Group Time Point Concentration (ng/mL) 1 NA 1 5.0567454 1 NA 2 14.4489166 1 NA 3 5.9581054 1 NA 4 9.324685 3 SED 1 1.2497753 3 SED 2 3.360509 3 SED 3 11.7027755 3 SED 4 7.1925056 4 SED 1 3.1228658 4 SED 2 2.7614501 4 SED 3 5.9036213 4 SED 4 5.2847588 5 SED 1 5.4514391 5 SED 2 2.9627867 5 SED 3 4.1411009 5 SED 4 3.2713928 6 SED 1 0.6919096 6 SED 2 0.446289 6 SED 3 -0.105794 6 SED 4 -2.2758182 7 SED 1 4.4438206 7 SED 2 8.6013436 7 SED 3 4.3131234 7 SED 4 4.4776216 9 SED 1 4.0932422 9 SED 2 13.3019162 9 SED 3 8.5672055 9 SED 4 4.911791 10 SED 1 2.7895052 10 SED 2 2.7284441 10 SED 3 1.00058 10 SED 4 3.7334768 11 P4 1 5.257298 11 P4 2 9.5027036 11 P4 3 3.1774098 11 P4 4 1.6360842 12 P4 1 0.9442904 12 P4 2 -0.5474604 12 P4 3 0.1353198 12 P4 4 1.7735416 13 P4 1 54.3769112 13 P4 2 47.159271 13 P4 3 51.7156458 13 P4 4 48.3400526 15 NA 1 25.9003064 15 NA 2 29.7933641 15 NA 3 28.7157182 15 NA 4 35.5281566 17 NA 1 0.1894014 17 NA 2 0.9059826 17 NA 3 4.5091692 17 NA 4 3.2878264 18 SED 1 1.572989 18 SED 2 3.094034 18 SED 3 0.0429304 18 SED 4 -0.105794 19 SED 1 16.231356 19 SED 2 14.4444098 19 SED 3 21.6372626 19 SED 4 21.8558424 20 SED 1 1.3994772 20 SED 2 0.4845968 20 SED 3 1.3318752 20 SED 4 -0.4032428 21 SED 1 2.6327267 21 SED 2 4.6213382 21 SED 3 4.598234 21 SED 4 2.5056536 23 SED 1 0.2810492 23 SED 2 1.6474976 23 SED 3 0.7266302 23 SED 4 0.9873776 25 SED 1 2.3026667 25 SED 2 3.228485 25 SED 3 5.9613818 25 SED 4 3.2466383 157

Figure 51. Comparison of Serum FST Concentrations between and within Groups.

Average serum FST concentration was not significantly different between the CON

(“NA”) walking group and the experimental groups; however, average serum FST concentration in the sedentary group was significantly less than in the P4 ACL group

(p<0.05). No within group differences were observed.

Discussion

Molecular indicators of the clinical benefits of exercise remain largely undetermined. In this study we have attempted to examine the potential beneficial effects of walking exercise on molecular and clinical measures of musculoskeletal health.

Additionally, we compared the effects of walking to another mode of exercise that included higher intensity, higher impact activity. Walking is prescribed for ease and accessibility to a general population; however, studies indicate that it may not be as 158

beneficial for increasing BMD and muscular strength as some other exercise protocols.

We have shown here that indeed, walking may not be the most beneficial mode of exercise for increasing bone and muscle strength. Further, we were not able to establish clear relationships between serum measures of FSTL3/FST/MSTN and clinical measures of bone and muscle strength.

We had observed an increase and elevation in serum FSTL3 concentration from baseline in our previous study in the mouse30 after 2, 7, and 15 days of walking exercise; however, these increases were observed within 6 hours of the exercise bout, and thus may represent a transient increase in serum FSTL3. While this transient increase may be physiologically relevant, the results obtained in the current study may better reflect the long term changes in FSTL3 concentration in response to walking. In this study, we did not observe significant changes in serum FSTL3 concentrations in response to any of the exercise interventions over time (Table 11, Figure 49). We expected FSTL3 levels to remain constant in the CON walking group because these individuals were continuously active and followed a training schedule to train for competitive walking events. We did expect to see an increase in FSTL3 concentration after the first week of the walking exercise in the SED group since we had observed the strongest increase in FSTL3 concentration in sedentary subjects at this timepoint previously;30 however, FSTL3 was not significantly elevated in these subjects after 7 days of walking. We may have observed an increase in serum FSTL3 in the P4ACL group if the additional training protocol we used here was a sufficiently strong stimulus over and above the exercise stimulus that this group was already receiving, as these individuals were already healthy

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and active. Interestingly, each individual appeared to have their own baseline levels of

FSTL3 that wavered little from that baseline in response to any of the exercise protocols.

The mechanism of regulation of these baseline levels in the serum is unknown and deserves further study.

One possible reason that the serum FSTL3 results in this study differed from our previous study was that we attempted to use a more quantitative approach to determine serum FSTL3 concentrations in this latest study. We used a human FLRG ELISA kit which has been tested extensively for specificity, precision, recovery, sensitivity, and linearity. The previous study used Western blotting to determine relative changes in

FSTL3 serum concentration. This methodological difference between the two studies may partially explain the conflicting results. Additionally, the previous study was performed in a lab under well controlled conditions: blood was drawn from subjects within 2-6 hours of exercising, and always on the specified days, which is optimal for assessment of myogenic gene expression changes, and likely also for serum clinical measures.305 In the current study, we were attempting to determine the clinical robustness of FSTL3 as a serum biomarker for BMD and muscle strength. As such, our volunteer subjects were requested to come to the lab for blood draws within a certain window of time, but as with any real-world situation, we had to accommodate our subjects’ work, school, and family schedules.

We also found that serum MSTN concentrations showed no consistent pattern over time within each subject (Table 12, Figure 50). As some studies indicate lower

MSTN levels in the muscle with training,306, 307 we hypothesized that the active groups

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(CON and P4ACL) would have lower serum MSTN levels while the SED group would have higher levels; however, we did not observe a significant pattern in this regard, although a trend in which the active groups had lower serum MSTN was noted.

However, it is possible that muscle MSTN expression levels do not correlate with serum levels, which could account for the results observed in this study. We also did not test for levels of ActRIIB, the MSTN receptor, which has been observed to be decreased with training200 and could account for the reduction in MSTN’s effects in some studies in response to exercise training. Other studies show an increase in MSTN levels after heavy resistance training.79 Nevertheless, further study on the MSTN response is warranted as studies on the response to MSTN after acute bouts of exercise versus long-term training remain inconclusive.308 It was somewhat surprising that serum FSTL3 and MSTN concentrations did not correlate, as FSTL3 is a binding partner of MSTN in serum,34, 309 although another study has also observed a lack of relationship between serum FSTL3 and MSTN.310 Regulation of MSTN by its antagonists may depend on the tissue, and the mechanisms remain unclear.62 In fact, it is believed that regulation of muscle mass by these mediators may occur largely at the tissue level through local actions versus circulating endocrine action.62 Thus, future studies should focus on muscle biopsies to determine the tissue-specific relationship between FSTL3 and MSTN in response to exercise. It appears that the FSTL3/MSTN relationship is not suitable for serum biomarker determination. It should be noted that we did not assay for serum levels of

GASP-1, the myostatin propeptide, or the glucocorticoid receptor, additional regulators of MSTN in the serum that could account for changes in MSTN levels,79, 311 as we

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wanted to specifically determine the relationship between MSTN and FSTL3 in response to walking exercise.

Finally, we determined serum concentrations of another MSTN binding partner,

FST (Table 13, Figure 51), that could potentially compensate for FSTL3 for some of its actions.62 Serum FST and MSTN levels showed no correlation, indicating that FST may not be affecting MSTN concentration in response to exercise in this study. However, there appeared to be large, but non-significant changes in FST levels over time within many individuals that was not related to the exercise interventions. The implication of these changes in regard to MSTN inhibition is unknown, but FST is known to cycle during the reproductive stages in the female,312 and IGF-1 may influence levels of FST313 indicating the complexity of the regulation of these molecules. There appeared to be a trend in that FST was higher in the younger women of the P4 group than of the women of the CON and SED groups (all were women except for one man in the SED group).

However, no age-specific trends in FST have been observed in a study that compared young (22-28 years) to older (65-92 years) women.314 It should be noted that the females in this study were 14, however, and lower in age than reported by other studies. In 2007,

Reame et al. showed an age-dependent decrease in FST,315 but Miyamoto et al. showed a positive correlation with age.316 An earlier study by Reame et al. also showed no differences in FST according to age.317 Finally, FST serum concentration in the present study was significantly lower in the SED group than in the P4ACL group. This result could be a reflection of higher FST in trained individuals, as FST is shown to increase in response to exercise.43, 318

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We next attempted to correlate serum FSTL3 concentrations with BMD data obtained from DXA analysis. The purpose of this exercise was to determine the suitability of FSTL3 as a biomarker for bone density. While DXA is the gold standard for determining BMD,319 it can be costly and time consuming in comparison to blood tests. A simple blood test for BMD would be ideal for not only diagnosis of low BMD, but convenient for determination of the effectiveness of an exercise program over time.

We did not show a positive correlation between FSTL3 and total, spine, or femur BMD in this study. We did observe a weak correlation between forearm BMD and FSTL3 concentration. Future experiments should determine a more suitable blood biomarker for

BMD.

The DXA results themselves did not show any significant changes in total body

BMD from baseline to post test in any of the three groups or across all sites (Table 10,

Figures 43-44). We compared the effects of two types of exercise on BMD. One was walking exercise, undertaken by previously sedentary subjects. The other was a training program that included core strengthening, neuromuscular training, and jumping/plyometric exercises; however, most of the subjects that enrolled in this training program were young, healthy, and already very physically active. These particular participants were primarily high school or college students who were playing one or more sports and who were either coming back from a previous ACL injury or wanted to prevent a first ACL injury. Likely because these subjects were so active at the outset, we did not observe a significant increase in BMD and FSTL3 concentration with training.

Perhaps the exercise stimulus administered in this study, although of higher intensity and

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impact than walking, was not sufficient for increasing BMD over and above the stimulus that these subjects were already receiving in their daily lives. Further, the P4 ACL exercises were performed at the OSU clinic three days a week for 45 minutes per session.

It is possible that a greater time commitment would have been required to see changes in

BMD with this protocol, as the actual time spent with weight bearing/ jumping exercises was only a portion of this exercise program. Although a walking program may be beneficial for increasing BMD, but it may need to be a longer intervention than the 6 weeks used here.320 Six weeks was chosen for this study because we wanted to observe the relationship between BMD and FSTL3 serum concentration at the early stages of a long-term exercise program, and to parallel the mouse studies. Loss of body mass could impact BMD; however, most subjects were instructed to maintain a consistent diet, and only two subjects lost weight (7 and 6 pounds). Finally, BMD at each site changed during the course of the study, suggesting that a redistribution of BMD could have occurred. While these changes were not significant, the trends bear further study.

Finally, muscular strength tests indicated that the walking exercise program was not consistently sufficient to increase muscular strength of the hip abductors, quadriceps, and hamstrings, although some significant differences were noted. This is unsurprising considering that the hip abductors are not strongly activated during walking exercise, and the walking stimulus was likely not great enough to induce changes in quadriceps and hamstring strength. Further, Biodex isokinetic dynamometry has some error associated with the technique.321, 322, 323, 324, 325 It is possible that our results are within the error level of the machine, and we are not able to observe significant differences. Likewise,

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the hip strength device used here has not been tested for test-retest reliability or error rate.

However, when significant improvements were noted, it was most often in the sedentary group. These results indicate that even a small stimulus such as walking can have an impact on muscular strength in a sedentary population.

It should be noted that the results of these experiments may not be generalizable to all populations as most of our subjects were female. The follistatins are reproductive hormones; therefore, the possibility of differential effects of these molecules on different sexes exists. This possibility deserves further study. The P4ACL group was biased in that they were mostly younger participants. Also, the P4ACL and CON group had less participants than the sedentary group, thus decreasing the possibility for finding truly significant effects in these groups. The P4ACL group consisted of young, athletic subjects, most of whom were already very active. It is possible that the exercise intervention used in this study was not a strong enough stimulus for this group to produce significant changes in bone and muscle measures. Nevertheless, this sample of convenience was used. The sedentary group was the most representative of the general population; however, it should be noted that these participants were not always completely sedentary, as some of them anecdotally mentioned various activities in which they participated. Additionally, many of them joined the study in the hopes of returning to exercise or of losing weight. While all subjects were instructed to maintain a consistent diet and not start other exercise activities during the study, some subjects clearly did not follow this advice during the entire 6 weeks. Any significant losses in lean mass in response to dieting could affect BMD results.326, 327

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The control walking group should have produced consistent measures since their workouts were expected to remain constant. However, the fact that some strength changes were observed may mean that they did change thir routines or that other factors are influencing the serum mediators and strength measures analyzed. The results may also be a factor of the error associated with the testing, as this change in strength was not consistently observed for all tests.

Conclusions

This work was intended to enhance fundamental understanding of the role of execise in molecular regulation of muscle and bone health by investigating a key pathway known to affect the musculoskeletal system in response to exercise. Determinaton of such pathways is important for development of objective clinical biomarkers of health and disease, as well as effectiveness of an exercise or physical therapy regimen. We found that FSTL3 levels collected at the timepoints tested does not represent a suitable serum biomarker surrogate for BMD in response to exercise, as it was not stably expressed over a long period of time, nor did it correlate to BMD measures. It would be beneficial from a clinical and training standpoint to determine in future studies an appropriate serum biomarker for bone and muscle health. Further, walking exercise is insufficient for increasing bone in human patients after six weeks, but it may provide some muscle strength gains in a sedentary population. For this reason, a greater exercise stimulus should be employed to provide the most advantageous bone and muscle building benefits to increase health.

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Chapter 6: Summary, Significance, and Future Work

In these studies we aimed to increase the available fundamental knowledge of molecular mechanisms of exercise. The study of exercise at the molecular level is important because knowledge of such mechanisms can lead to new advances in maintaining health and novel strategies to fight or prevent diseases. To study these molecular mechanisms, we used cell, mouse and rat models as well as human subjects with the goal of increasing understanding on both a basic science and a translational level.

We first examined gene expression changes underlying cartilage homeostasis in normal, healthy tissue. Microarray analysis was used to determine changes in gene expression in response to the beginning of a low intensity walking exercise program.

One of the major findings of this study is that mechanical signals generated by exercise elicited a regenerative response in the cartilage that increases ECM synthesis and cellular metabolism to promote health and homeostasis of the tissue. One of the major effects of exercise on cartilage is the maintenance of the cartilage ECM and the cartilage phenotype by inducing the expression of cartilage associated ECM genes while downregulating non- articular cartilage associated ECM genes and genes for muscle and bone. Upregulation of genes for growth factors further supported the regeneration of the ECM. Exercise maintained health of the chondrocytes and ECM by upregulating cell metabolism and cytoskeletal components to support cellular biomechanics and nutrient uptake. For 167

example, genes involved in cell surface mediated signaling and signal transduction, adhesion, and cell growth/differentiation were upregulated to support cell metabolism, while genes encoding microtubules and intermediate filaments were upregulated to support the cytoskeleton.

Importantly, the results of this study provided a potential mechanism for the beneficial effects of exercise on preventing onset and progression of OA. For example, exercise suppressed the chondrocyte division and proliferation observed during the onset of OA. Although exercise clearly exerts regenerative influences on cartilage, it inhibited several genes integrally associated with cell cycle progression and mitosis. Additionally, exercise regulates the immune response such that many anti-inflammatory genes are upregulated and many pro-inflammatory genes, and several genes expressed in OA are downregulated. Furthermore, many of the genes encoding the collagenases and aggrecanases that are detrimental to cartilage integrity are significantly inhibited by exercise, while genes that code for protease inhibitors are upregulated to minimize cartilage proteolysis.

Overall, this study provided information on the role of exercise in maintaining cartilage health by maintaining cartilage matrix homeostasis, suppressing cell proliferation signals that lead to the onset of OA, and regulating inflammatory processes known to be active in OA; however, microarray studies are usually a first step in a process. Some of the identified genes could be studied more closely, as there were certainly novel proteins identified through the microarray analysis for which a definitive function could not be identified through typical published sources and databases

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(PubMed, GeneCards, etc.). Once these proteins have been identified, use of transgenic mouse models could then be used to determine protein function. Microarray analyses in general can be used to compare gene expression in two or more experimental conditions in both animals and humans. For example, gene expression in an animal model of injury could be compared to that of an uninjured control animal.

Our microarray analysis was limited to 15 days of exercise. The effect of a long term exercise program on gene expression in cartilage is certainly warranted, as is a comparison of different modes, intensities, and durations of each exercise bout. Low impact walking was used in this study to determine gene expression consequences of exercise training. The effects of high impact, high intensity exercise on gene expression should also be studied. Further, the exercise in this study was 45 min/day for 15 days; the consequences of longer exercise bouts or differing rest intervals between the exercise bouts should be determined.

One of the genes shown by our microarray analysis to be upregulated by more than 1.5 fold in the cartilage was FSTL3, which was confirmed through quantitative PCR measurements. FSTL3 is a potential mediator linking bone and muscle health.

Mechanisms of exercise mediated bone and muscle coordination remain largely undetermined, and we aimed to further define a potential mechanim involving FSTL3.

Through this study, we attempted to determine the effects of walking-mediated upregulation of FSTL3, a known regulator of bone growth, on muscle strength through its actions on MSTN. FSTL3 binds to MSTN to prevent its downstream actions including decreased muscular hypertrophy and increased muscular atrophy. In spite of its

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challenges, MSTN has been regarded as an attractive target for the development of therapeutics to increase muscle growth through its inhibition.

Using transgenic mouse models, we did not observe an effect of the loss of

FSTL3 on the muscular strength of our animals, likely because we were unable to produce a sufficient training effect using the walking exercise protcol. Although a previous study showed that this protocol was sufficient to produce bone growth in the mice, it was not sufficiently rigorous to produce muscle hypertrophy. The effects of other exercise protocols capable of increasing muscle strength in mice, including resistance and high intensity exercise, on mouse FSTL3 levels should be examined.

Mouse muscle weight to body weight ratios confirmed no change in muscle hypertrophy in response to MSTN inhibition by FSTL3. Using a cell culture system, we also did not observe an increase in myotube hypertrophy in response to FSTL3 overexpression or recombinant FSTL3 treatment of C2C12 cells, although experimental limitations may have precluded genuine outcomes.

Although we limited our studies to follistatin family members, other MSTN mediators also should be tested for potential effects on muscle hypertrophy. We did attempt to determine whether FST and FSTL3 could compensate for one another to inhibit MSTN. Our results indicated that some compensation between these molecules may occur, although the small sample size of the DKO group may have impacted these results. Studies show conflicting results regarding the effectiveness of MSTN inhibition to increase muscle growth and strength. Heterogeneity of studies in terms of animal models used, time course of treatment and/or sampling, and treatment strength all

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complicate interpretation of experimental results and make comparisons between studies difficult. It is clear that much is still unknown about the most effective way to inhibit

MSTN to produce the best gains in muscle.

Although we were ultimately unsuccessful at increasing muscle hypertrophy through MSTN inhibition by FSTL3, this study provides information on the effectiveness of walking exercise in the mouse to promote bone and muscle cross-talk through the

MSTN/FSTL3 mediators. It is clear that additional experiments to determine molecular mechanisms of exercise are needed. In terms of the FTL3/MSTN/FST system in particular, serum and tissue concentrations of these mediators both at rest and in response to exercise should be determined to better refine their potential compensatory roles.

Future studies should determine a more effective exercise protocol for improving bone and muscle health, including other exercise modes, intensities, and durations to increase

FSTL3 serum concentration/bone growth/muscle force. Circulating mediators are becoming increasing studied in order to determine how muscle and bone communicate.

Other mechanisms of bone and muscle crosstalk should also be identified. Mechanisms mediating strength of bone and muscle in response to exercise are likely collectively regulated.

We intended for this study to be a first step in the process for optimizing a MSTN inhibitor, FSTL3, as a clinical therapeutic. However, we learned that FSTL3 may not be the best choice for clinical development, and that MSTN inhibition is complicated by various factors. For example, the effects of MSTN inhibition on muscle hypertrophy and/or hyperplasia should be clarified. The effects of MSTN inhibition on muscle

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function versus size alone also should be further investigated. Finally, we only examined the effects of FSTL3 on MSTN; however, there are other MSTN binding proteins and regulatory proteins that could also regulate hypertrophy. For example, GASP-1 and

SMURF1 have also been shown to be upregulated in response to exercise. Refinement of

MSTN inhibition could allow for better therapeutics to increase muscle strength.

In addition to skeletal muscle, we also examined cardiac muscle to determine the effects of the follistatin family molecules in regulating heart size and function. Although the follistatin family members share some structural similarities, these molecules have been shown to have different functions. Further, while the actions of FSTL1 and FSTL3 have been somewhat characterized in the heart, studies on the role of the FST315 isoform are lacking. In this experiment, both wild-type and FST288 mice were subjected to transverse aortic constriction (TAC), a method of producing pressure overload induced hypertrophy and eventual heart failure, for 4 weeks. After the 4 weeks, WT mouse hearts were significantly hypertrophied in comparison to sham control mice, whereas the hypertrophic response was blunted entirely in the FST288 mice. This is the first study, to our knowledge, to determine the response of the heart to altered FST signaling. Western blotting results indicated that the hypertrophic response was blunted in the FST288 mice due to altered pAKT and pGSK growth pathway signaling. Although WT mice subjected to TAC showed an increased expression of this pathway, FST288 TAC mice showed a decrease in pAKT signaling. We concluded that loss of the FST315 isoform abolishes the cardiac hypertrophy observed after TAC-induced pressure overload. We found that

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this effect is a result of reduced AKT pathway signaling, and that the FST isoform

FST315 is one contributor to pathological cardiac hypertrophy.

Mechanisms of heart remodeling and failure are important to determine, as this is a significant health problem in the U.S. An understanding of the factors that lead to pathological hypertrophy in the heart could lead to the development of new therapies to treat heart failure. The presence of secreted factors in the heart that contribute to pathological hypertrophy and cardiac remodeling are of recent interest, and new factors should be identified and investigated. As an example, failing hearts could be examined to determine what proteins are expressed as compared to control hearts. Such an approach was used to find the upregulation of FSTL1 in Akt transgenic mice.197 The contributions of FST288 and FST315 to other pathways involved in heart failure, such as fibrosis, also should be examined.242

Finally, we determined the effects of FSTL3 on bone and muscle in a human population. Although in vitro and in vivo experiments allowed us to learn more about mechanistic effects of circulating mediators, we wanted to determine the translational potential of these molecules and how they could affect human health. Molecular indicators of the clinical benefits of exercise remain largely undetermined. In this study we attempted to examine the potential beneficial effects of walking exercise on molecular and clinical measures of musculoskeletal health. Additionally, we compared the effects of walking to another mode of exercise that included higher intensity, higher impact activity. Walking is prescribed for ease and accessibility to a general population; however, studies indicate that it may not be as beneficial for increasing BMD and

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muscular strength as some other exercise protocols that impart a higher impact stimulus or longer term stimulus. We have shown here that indeed, walking may not be the most beneficial mode of exercise for increasing bone and muscle strength. Further, we were not able to establish clear relationships between serum measures of FSTL3/FST/MSTN and clinical measures of bone and muscle strength.

Each individual appeared to have their own baseline levels of FSTL3 that wavered little from that baseline in response to any of the exercise protocols. The mechanism of regulation of these baseline levels in the serum is unknown and deserves further study. A more consistent approach to the analysis of FSTL3 levels in serum is warranted, as ELISA and Western blotting approaches may provide different results. The follistatins are reproductive hormones; therefore, the possibility of differential effects of these molecules on different sexes exists. This possibility deserves further study.

We attempted to correlate serum FSTL3 concentrations with BMD data obtained from DXA analysis. The purpose of this exercise was to determine the suitability of

FSTL3 as a biomarker for bone density, but we did not find a correlation between FSTL3 and total, spine, or femur BMD in this study. Reliable serum markers of BMD and muscle strength should be determined. Such biomarkers would be helpful in designing appropriate exercise interventions in both healthy and diseased states.

The mechanisms of MSTN regulation in response to exercise should be further defined. Some studies have shown lower MSTN in the muscles with exercise, while others report no change. It is possible that muscle MSTN expression levels do not correlate with serum levels, or that regulators other than FSTL3 are affecting MSTN

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levels in response to exercise. Such possibilities should be investigated, as we observed no relationship between serum FSTL3 and MSTN levels in this experiment. Future studies should focus on muscle biopsies to determine the tissue-specific relationship between FSTL3 and MSTN in response to exercise. Furthermore, studies on the response of MSTN after acute bouts versus long-term exercise training also need to be conducted.

This translational study was intended to investigate the molecular regulation of bone and muscle health in response to exercise. Determinaton of such regulation is important for development of objective clinical biomarkers of health and disease, as well as effectiveness of an exercise or physical therapy program. Although FSTL3 was not found to be a suitable serum biomarker for BMD in response to exercise in this study, future studies should determine appropriate serum biomarkers for bone and muscle health. This study also provides information on the effectiveness of walking to increase bone and muscle strength in humans. A greater exercise stimulus may need to be applied in order to provide the most advantageous bone and muscle building benefits to increase health.

While we were ultimately unsuccessful in finding a serum biomarker for BMD and muscle strength in FSTL3, this culmination of this work adds to a body of knowledge of molecular mechanisms of exercise. In these studies, we examined potential mechanisms on the organismal as well as specific systems level, including the musculoskeletal system and heart, and by using cell and animal models. We also used human subjects to determine the translational potential of our work. This approach should be used for future molecular exercise research, as exercise integrates multiple

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systems. The ability to exploit molecular indicators of exercise effectiveness as well as discover novel therapeutic options are clear advantages to studying molecular exercise physiology, as these mechanisms could help to improve upon preventative healthcare and reduce healthcare cost burden in our country.

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