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Isosorbide Dinitrate and L-Arginine

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Title:
Isosorbide Dinitrate and L-Arginine Fountain of Youth for Aged Muscle Regeneration?
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1 online resource (48 p.)
Language:
english
Creator:
Miller, Kevin J
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Applied Physiology and Kinesiology
Committee Chair:
Criswell, David S
Committee Members:
Powers, Scotty K
Dodd, Stephen

Subjects

Subjects / Keywords:
arginine -- isdn -- muscle -- myogenesis -- nitrate -- nitric -- no -- oxide -- regeneration -- sarcopenia -- satellite
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre:
Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Senescent myofibers generally are characterized by a regenerative deficit in comparison to their younger counterparts, one of many symptoms of sarcopenia. This deficit is largely due to the blunting of satellite cell activation, likely caused by systemic factors. In response to injury, nitric oxide is produced and is one of the primary mediators of the subsequent regenerative response. This response entails activation of matrix metalloproteinases, endopeptidases that cleave hepatic growth factor from its extracellular binding site and result in satellite cell activation. It has been well established that L-arginine and isosorbide dinitrate are both potent precursors to NO, and the purpose of our investigation was to determine if either NO donor, enzymatic or non-enzymatic respectively, had any impact in rescuing the myogenic capacity of the senescent muscle. We hypothesized that both supplements would have a positive effect on the regeneration of the treated aged subjects compared to their senescent control counterparts. Each subgroup of mice consisted of four 23-month-old individuals, with four young controls to serve as an index of myogenic rescue. The animals were supplemented with the treatment diets for a week prior to cardiotoxin injection, then sacrificed at either five or ten days post injection. Histochemical analysis was used to determine extent of regeneration. Our results indicated a recovery of early regenerative capacity with both supplements at five days, but not at ten days. We conclude that while promising, further investigation is necessary to determine the true efficacy of these supplements for the treatment and prevention of sarcopenia.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Kevin J Miller.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Criswell, David S.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045998:00001

MISSING IMAGE

Material Information

Title:
Isosorbide Dinitrate and L-Arginine Fountain of Youth for Aged Muscle Regeneration?
Physical Description:
1 online resource (48 p.)
Language:
english
Creator:
Miller, Kevin J
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Applied Physiology and Kinesiology
Committee Chair:
Criswell, David S
Committee Members:
Powers, Scotty K
Dodd, Stephen

Subjects

Subjects / Keywords:
arginine -- isdn -- muscle -- myogenesis -- nitrate -- nitric -- no -- oxide -- regeneration -- sarcopenia -- satellite
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre:
Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Senescent myofibers generally are characterized by a regenerative deficit in comparison to their younger counterparts, one of many symptoms of sarcopenia. This deficit is largely due to the blunting of satellite cell activation, likely caused by systemic factors. In response to injury, nitric oxide is produced and is one of the primary mediators of the subsequent regenerative response. This response entails activation of matrix metalloproteinases, endopeptidases that cleave hepatic growth factor from its extracellular binding site and result in satellite cell activation. It has been well established that L-arginine and isosorbide dinitrate are both potent precursors to NO, and the purpose of our investigation was to determine if either NO donor, enzymatic or non-enzymatic respectively, had any impact in rescuing the myogenic capacity of the senescent muscle. We hypothesized that both supplements would have a positive effect on the regeneration of the treated aged subjects compared to their senescent control counterparts. Each subgroup of mice consisted of four 23-month-old individuals, with four young controls to serve as an index of myogenic rescue. The animals were supplemented with the treatment diets for a week prior to cardiotoxin injection, then sacrificed at either five or ten days post injection. Histochemical analysis was used to determine extent of regeneration. Our results indicated a recovery of early regenerative capacity with both supplements at five days, but not at ten days. We conclude that while promising, further investigation is necessary to determine the true efficacy of these supplements for the treatment and prevention of sarcopenia.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Kevin J Miller.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Criswell, David S.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045998:00001


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1 ISOSORBIDE DINITRATE AND L ARGININE : FOUNTAIN OF YOUTH FOR AGED MUSCLE REGENERATION? By KEVIN JAMES MILLER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Kevin James Miller

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3 To my parents to whom I owe everything

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4 ACKNOWLEDGEMENTS First, I would be remiss if I did not thank my advisor, Dr. David Criswell, for his unwavering guidance and patience with me as I learned how to do science from scratch. There were many days in the beginning where I had difficulties with even the most basi c of scientific tasks, yet he stuck with me and invested copious amounts of his time and energy into training me and making this all possible. Second, I would also like to thank my committee members, Drs. Scott Powers and Steve Dodd, for their inspiration al teaching that encouraged me to pursue this degree path. Additionally, I would like to thank one of my undergraduate professors and the graduate advisor, Dr. Chris Janelle, for investing a large amount of his time and energy into giving me the guidance n Finally, I thank my parents, who have made this all possible.

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5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Backgro und ................................ ................................ ................................ ............. 12 Specific Aims and Hypotheses ................................ ................................ ............... 15 Strengths and Limitations ................................ ................................ ....................... 16 Clinical Significance ................................ ................................ ................................ 17 2 LITERATURE REVIEW ................................ ................................ .......................... 19 Foreword ................................ ................................ ................................ ................. 19 Overview of Myogenesis ................................ ................................ ......................... 19 Satellite Cell Delineation ................................ ................................ ................... 20 The First Stage: Degeneration and Inflammation ................................ ............. 21 The Second Stage: Regeneration ................................ ................................ .... 21 Hanging in the Balance: Regeneration and Fibrosis ................................ ........ 22 Get ting to the Source of Age Related Deficits ................................ ........................ 24 L Arginine and ISDN ................................ ................................ ............................... 26 Conclusion ................................ ................................ ................................ .............. 28 3 METHODS ................................ ................................ ................................ .............. 29 Experimental Design ................................ ................................ ............................... 29 Animals ................................ ................................ ................................ ............. 29 Experimental Diets ................................ ................................ ........................... 30 Experimental Protocols ................................ ................................ ........................... 30 Cardiotoxin Injury ................................ ................................ ............................. 30 Tissue Removal and Preparation ................................ ................................ ..... 30 Hematoxylin & Eosin Stain ................................ ................................ ............... 31 Analyses ................................ ................................ ................................ ................. 31 Image Analyses ................................ ................................ ................................ 31 Statistical Analyses ................................ ................................ .......................... 32

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6 4 RESULTS ................................ ................................ ................................ ............... 33 NO Augm enting Supplements as a Method of Nullifying Age Associated Muscle Wasting ................................ ................................ ................................ ................ 33 L Arginine Supplementation Increases the Regenerative Response of Senescent Muscle at Five Days Post Injury ................................ ......................... 33 ISDN Supplementation Shows a Positive Trend Toward Increasing the Myogenic Capacity of Senescent Muscle ................................ ............................ 38 5 DISCUSSION ................................ ................................ ................................ ......... 40 Principal Findings ................................ ................................ ................................ ... 40 Matrix Metalloproteinases are Key Modulators of Satellite Cell Function ............... 41 NO Supplementaion as a Means of Sarcopenia Treatment ................................ .... 41 P ossible Side Effects of L Arginine ................................ ................................ ......... 42 Future Directions ................................ ................................ ................................ .... 43 Conclusions ................................ ................................ ................................ ............ 44 LIST OF REFERENCES ................................ ................................ ............................... 45 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 48

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7 LIST OF TABLES Table page 3 1 Experimental Design ................................ ................................ .......................... 2 9 4 1 Average tibialis anterior (TA) and body masses (BM) for all groups. Values are mean SEM. ................................ ................................ ................................ .. 33 4 2 Recorded and calculated parameters of regeneration for all samples. ............... 34

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8 LIST OF FIGURES Figure page 2 1 Overview of Notch/Delta signaling ................................ ................................ ...... 24 4 1 Representative H&E sections from all groups. TA muscle was removed at 5 or 10 days and analyzed using ImageJ. ................................ ............................. 35 4 2 Average TA cross sectional area post CTX injection. *Significantly different from YC (p<0.05). ................................ ................................ .............................. 36 4 3 Regeneration Index (RI) of all groups post CTX injection. # Significantly different from YC (p<0.05). *Significantly d ifferent from OC (p<0.05). ............... 37 4 4 Total fiber area for all groups post CTX injection. ................................ .............. 38

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9 LIST OF ABBREVIATIONS A NOVA Analysis of variance CL Control CTX Cardiotoxin H&E Hematoxylin and e osin HGF Hepatic growth factor ISDN Isosorbide d initrate L NAME N nitro l arginine methyl ester M DX Mouse muscular dystrophy model MMP Matrix metalloproteinase MRF Muscle regulatory factor M YF 5 Myogenic factor five M YO D Myogenic differentiation antigen NO Nitric oxide N NOS Neuronal nitric oxide synthase NOS N itric oxide synthase OA Old l arginine supplemented mice OC Old control supplemented mic e OCT Optimal cutting temperature compound OI Old isorbide dintrate supplemented mice OPN Osteopontin RI Regeneration index TA Tibialis anterior TACXA Tibialis anterior cross sectional area TGF Tumor growth factor beta

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ISOSORBIDE DINITRATE AND L ARGININE: FOUNTAIN OF YOUTH FOR AGED MUSCLE REGENERATION? By Kevin Jam es Miller August 2013 Chair: David Criswell Major: Applied Physiology and Kinesiology Senescent myofibers generally are characterized by a regenerative deficit in comparison to their younger counterparts one of many symptoms of sarcopenia. This deficit is largely due to the blunting of satellite cell activation, likely caused by systemic factors. In response to injury, nitric oxide is produced and is one of the primary mediators of the subsequent regenerative response. This response entails activation of matri x metalloproteinases, endopeptidases that cleave hepatic growth factor from its extracellular binding site and result in satellite cell activation. It has been well es tablished that L arginine and i sosorbide dinitrate are both potent pr ecursors to NO, and the purpose of our investigation was to determine if either NO donor enzymatic or non enzymatic respectively, had any impact in rescuing the myogenic capacity of the senescent muscle. We hypothesized that both supplements would have a positive effect on the regeneration of the treated aged subjects compared to their senescent control counterparts. Each sub group of mice consisted of four 23 month old individuals, with four young controls to serve as an index of myogenic rescue The anima ls were supplemented with the treatment diets for a week prior to cardiotoxin injection, then sacrificed at either five or ten days post injection. Histochemical analysis was used to

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11 determine extent of regeneration. Our results indicated a recovery of ear ly regenerative capacity with both supplements at five days, but not at ten days. We conclude that while promising, further investigation is necessary to determine the true efficacy of these supplements for the treatment and prevention of sarcopenia.

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12 CHAPTER 1 INTRODUCTION Background The importance of understanding the decline of the muscular system as we age is of fundamental importance to human health and wellness. The process of aging in humans has a multitude of effects on the body. However, barring overt disease, the loss of muscl e strength and mass with advancing age, termed sarcopenia, and decreased muscular regenerative ability has the greatest impact on quality of life, producing the classic senescent phenotype often referred to as frailty. Sarcopenia and reduced regenerative p time passes. Nitric oxide (NO), a molecule with seemingly unbounded physiological influence, has been implicated in possible attenuation of these processes due to its involvement in several muscle regeneration pathways, including satellite cell activation, proliferation, and protein synthesis. It is generally accepted that muscle trauma and recovery occurs in three phases: degeneration, regeneration, and reconstruction. NO has bee n shown to exert some level of control over each of these three phases (17) and may be altered with aging. Therefore, augmentation of NO production or bioavailability may provide a convenient target for pharma cological or nutritional amelioration of age related deficits in the muscle regeneration process. Muscle regeneration is a complex process of vital importance in mammalian organisms, controlled through a myriad of pathways. Of utmost significance to this p rocess is the pathway of satellite cell activation and proliferation. Satellite cells are progenitor cells located on the muscle fiber that are quiescent until a sufficient stimulus is received to initiate their activation. It is at this crucial juncture t hat the satellite cells

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13 undergo proliferation and myogenic differentiation, completing the transition to mature muscle cells. This differentiation is largely controlled by two myogenic regulatory factors, MyoD and Myf 5. MyoD has been implicated in the progression to compl ete differentiation, whereas Myf 5 is thought to be involved with renewal of the satellite cell pool (9) It is well established that satellite cell populatio ns in aging muscle are resistant to activation and proliferation, and this is thought to be a primary cause of reduced regenerative potential in aging muscle. This resistance to activation and proliferation was thought to be an intrinsic property of the ag ing satellite cell until research from Rando and colleagues over the last decade proved otherwise. It is now generally accepted that aging muscle retains a robust regenerative potential if the resident satellite cells are exposed to the proper environment. Specifically, exposure of satellite cells from aging mouse muscle to serum from young mice, either in vitro or in vivo restores satellite cell activity in injured muscle and functional recovery of the muscle (12) Ultimately an understanding of the positive and negative regulators of satellite cell activation and proliferation could lead to nutritional or pharmacological methods of rescuing the regenerative potential of aging muscle. Nitric oxide is a near omnipresent molecule in the human body, as almost every cell possessing a nucleus contains one of the three NOS isoforms. In addition, it performs a variety of functions such as neural signal conduction, vasodilation, and san itation (5) Recent evidence supports the idea that nitric oxide also exerts regulatory control over muscle regeneration. It has been shown to have a role in every phase of muscle response to injury and exudes control over an immense cascade o f reactions, many of which are yet to be discovered. The activation of satellite cells is one of the first

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14 and most important responses to muscle injury, and is initiated by hepatocyte growth factor (HGF) binding to the c met receptor on quiescent satellit e cells. It is now known that nitric oxide controls this process. Muscular NO production is induced through a mechanical stretch stimulus which activa tes nitric oxide synthase (NOS) and proceeds to induce activation of matrix metalloproteinases (MMPs). MMP s have been shown to proteolyticly release HGF from an extracellular tether, allowing it to quickly activate neighboring satellite cells. The necessity of NO production for skeletal muscle regeneration was shown by Filippin et al. (2011) who demonstrated that inhibition of NOS activity prevents normal regeneration of skeletal muscle following crush injury in young mice. It has recently been shown that supplemental nitric oxide in vitro, via addition of a nitric oxide donor, can augment satellite cell activ ation on mechanically stimulated isolated myofibers from old mice (17) Therefore, it is reasonable to hypothesize that supplementation of endogenous NO production in vivo may overcome the aging deficit in sate llite cell activity following muscle injury. L arginine is an amino acid that serves a plethora of roles within the body. It is a primary intermediate in the Urea cycle and is a major constituent in the prod uction of polyamines as well as glutamate and c reatine (24) However, quite possibly its most important physiological role relative to skeletal muscle regeneration is that of precursor to NO production. Through the action of the enzyme NOS, L arginine is converted to NO and citrulline. L arginine supplementation has been shown to augment wound healing, presumably via NO related stimulation of angiogenesis. Only one study has e xamined L arginine and skeletal muscl e regeneration, reporting that L arginine

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15 supplementation significantly improved regeneration in a mouse model involving damage to both skeletal muscle fibers and muscle vasculature (25) It is unknown whether this effect was due to NO action on satellite cells or angiogenesis. Isosorbide Dinitrate (ISDN) is a known vasodilatory agent used in the treatment of hear t disease, as well as an NO donor. ISDN has been shown to enhance skeletal muscle regeneration in a mouse model of muscular dystrophy. ISDN has a high potential for clinical efficacy because of a general lack of side effects (22) Further, it does not rely on the action of the NOS enzyme to increase NO availability and should, therefore, retain its therapeutic value even in situations where the N OS enzyme is down regulated. Aging skeletal muscle exhibits a significant impairment of regenerative potential, due primarily to inhibition of satellite cell activity by extrinsic factors in the aging environment. The rescue of regenerative potential in a ging muscle by exposure of the muscle to serum from a young animal suggests the possibility of successful pharmacological or nutritional strategies to stimulate regeneration in aging muscle. Nitric oxide is a key factor in the activation of satellite cells following muscle injury, and exposure of isolated myofibers from old mice to L arginine or an NO donor ameliorates the aging deficit in mechanical activation of satellite cells (4) There fore, the following aims and hypotheses were addressed in this study. Specific Aims and Hypotheses Specific Aim: To investigate whether elevated levels of nitric oxide achieved through exogenous supplementation will have positive effects on muscle regenera tion in aged mice.

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16 Sub Aim 1: To determine if administration of ISDN will elicit positive changes in muscle regeneration in aged subjects. Rationale: ISDN is a chemical compound that is widely used in the field of cardiology as a vasodilatory agent, causin g the vasculature to expand and increase blood flow. These properties can be attributed to this compound as a result of its status as an NO donor, and it has been shown to improve muscle regeneration in muscular dystrophic mice. Hypothesis 1: Exogenous su pplementation of ISDN will result in a greater extent of muscle regeneration response to injury in aging subjects (22) Sub Aim 2: To determine if augmented amounts of L arginine will increase the regenerative response to injury in aging muscle. Rationale: L arginine is a naturally occurring amino acid that is produced by the body and utilized in a variety of physiological functions, ranging from the urea cycle to the production of NO. It is this role as a precursor to NO that warrants the investigation of i ts possible effects on muscle regeneration. Additionally, L arginine has been shown to increase muscle regeneration in mice with induced muscle injury, a promising development (25) Hypothesis 2: L arginine supplementation will increase the regenerative response to muscle injury in aging subjects. Strengths and Limitations The principal strength of this in vivo study is the practical clinical signif icance afforded by doing a whole animal model of muscle injury. This is in direct contrast to an in vitro study, where the isolation of cells outside the animal can lead to potentially unrealistic results. In addition, the model chosen enables a more accur ate determination

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17 of the inherent clinical efficacy of the substances chosen. This is also the first study to investigate the benefits of L arginine and ISDN supplementation on aged muscle response to injury. This research design is limited in its ability to determine the mechanism of potential effects of the dietary treatments on muscle regeneration. Nitric oxide signaling can affect a myriad of events in the intact animal including satellite cell activity, muscle blood flow, protein synthesis, and mitocho ndrial respiration. Effects of L arginine and ISDN could result from any of these or other changes. Therefore, future studies will be required to determine the cellular mechanisms underlying our results. Other limitations include the use of the mouse as a model of human physiology and restriction of observations to male mice at 3 and 24 months of age. Clinical Significance The loss of skeletal muscle regenerative potential with aging is a serious clinical problem. Age related declines in overall muscular s trength, balance, joint mobility, and visual and hearing acuity lead to increased chances of falls and orthopedic injury. Unfortunately, elderly patients may never fully regain muscular strength following an injury, due to reduced muscle regenerative poten tial. Therefore, a muscle injury can invoke a cycle of physical decline into frailty characterized by loss of independence and a dramatic reduction in quality of life. The recent recognition that aging skeletal muscle is fully capable of robust regeneratio n if exposed to the proper environment suggests that nutritional or pharmacological interventions could rescue the regenerative potential of aging skeletal muscle. Based on the known role of nitric oxide in muscle regeneration, and previously published rep orts of NO donors and L arginine effects on isolated myofibers from aging mice, we have tested the efficacy of L arginine and ISDN

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18 supplementation in an in vivo model of muscle injury in aging mice. If effective, these supplements could have immediate impa ct on the health and wellness of elderly individuals.

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19 CHAPTER 2 LITERATURE REVIEW Foreword The recent finding that skeletal muscle in aged individuals can exhibit a near complete regenerative response when exposed to young serum has profound implica tions on the realm of sarcopenia research (13) This discovery clearly indicates there are some unknown factors in the adolescent serum that are allowing for significantly increased recuperation that are either degraded or simply absent in the aged environment. Therefore, we investigated the potential of supplements such as L arginine or ISDN to attenuate such decreases in aged animals in hopes of providing a model that could be extrapolated to the proble m of sarcopenia in humans. Overview of Myogenesis Myogenesis is a very complex and multi faceted process, from muscle degeneration and inflammation, to satellite cell activation and proliferation. Myogenesis begins with an injury to the muscle, mechanical or otherwise, that damages the myofibers and causes an inflammatory response. This results in activation and proliferation of satellite cells, muscular stem cells whose role and characteristics will be thoroughly discussed later in this review. These cells are then directed by chemotaxis to the site of myotrauma, where they either fuse to the existing fiber and create a hypertrophic response, or fuse with other satellite cells to form new myotubes, a hyperplasic response. In both cases a fraction of the new cells return to quiescence and myogenesis is complete. Therefore, the satellite cell, being responsible for such a large portion of the regenerative process, holds the key to

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20 understanding and attenuating the factors res ponsible for declining muscle function in the aging population. Satellite Cell Delineation Satellite cells are progenitor cells situated between the basal lamina and the myofibers and are derived during embryonic development from the dermomyotome, an epith elial like derivative of early somites that surround the notochord. Recent work by Gros et al. has reaffirmed the status of satellite cells as exclusively somatic and not hematopoietic in origin (18) It has be en long established that satellite cells are legitimate stem cells, meeting the requirements of progeny and self renewal. These properties are what makes them so valuable, and have generated immense scientific interest in their possible therapeutic applica tions. In addition, recent research has shown their differentiation potential is not limited merely to skeletal muscle, but can be extended to adipocytes and osteocytes when exposed to the correct growth factors (2) This exciting discovery is not without its flaws, however, as cross contamination from non myogenic cells is a concern. Furthermore, satellite cells have been purported to not only be multipotent, but heterogenic as well, even between those cells inhabiting the same myofibers. Satellite cells in any given fiber can differ in their differentiation capacity, specificity, and ability to proliferate and regenerate (26) However, despite our knowledge of their heterogeneity, many questions remain, not the l east of which are in regard to age related changes. It has been demonstrated that satellite cells do indeed lose efficacy in aged populations, likely due to a cascade of systemic effects in the circulation as well as overall decreases in number and functio n (6) Therefore, elucidating the factors responsible for the deterioration of the regenerative environment

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21 with age and its corresponding ro le in satellite cell activity and efficacy is a primary focus within the study of myogenesis, and more specifically, muscle regeneration. The First Stage: Degeneration and Inflammation The first component of muscle regeneration is degeneration and inflamma tion of the myofibers. In exercise or other injurious stimuli, muscle fibers are damaged and become necrotic. These insults damage not only the structural proteins of the fiber itself, but the resulting transient increase in sarcolemmal membrane permeabili ty enhances plasmalemma also results in secretion of muscle proteins such as creatine kinase and troponin I into the blood, which explains why these molecules are the most commonly used biomarkers of muscle injury, especially in conditions such as muscular dystrophy and myocardial infarction (29) (30) Concomitant to the protein release, inflammatory cells are directed by chemotaxis to the site of muscle injury, with the first responders being neutrophils, increasing in as little as one hour after damage (23) Following the inf iltration of the neutrophils, macrophages travel to the necrotic area through the vasculature and reach their peak about 2 days post injury. Not only do these cells clear the area of compromised sarcolemmal, contractile, and other tissue, in vitro research has implicated them in activation and chemoattraction of satellite cells (8) At this point, regeneration of the myofiber can begin and satellite cells play a n integral role in the process. The Second Stage: Regeneration Muscle regeneration is largely mediated by the activity of satellite cell. Nitric oxide, acting in an enabler role, increases the activity of matrix metalloproteinases gmented expression of hepatic growth factor (HGF), widely

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22 considered an integral activa tor of the satellite cell Upon the first few hours of the upregulated, causing sa tellite cells to progress to the myoblast stage, where rapid proliferation occurs. Myoblasts then withdraw from the cell cycle and express the late myoblasts fail to differentiate and instead reprise their role as quiescent satellite cells, thought to serve the purpose of self renewal. The differentiated myocytes then merge contracting muscle fibers (9) nothing of the balance between regeneration and fibrosis. Hanging in the Balance: Regeneration and Fibrosis The degr ee of muscle healing has been defined as the balance between regeneration and fibrosis. The main fibrotic unit is the fibroblast, accompanied by collagen types I and III, as well as glycoproteins and proteoglycans. The fibroblast has the ability to indefin itely promote the inflammatory response, and despite the aforementioned mitogenic characteristics of macrophages, excessive amounts of certain secreted factors such as transforming growth factor beta 1 (TGF satellite cell differentiation and as a result attenuate myogenesis as a whole (7) TGF has been shown to be culpable in the in scar formation during myogenesis, stimulating the synthesis of extracellular matrix proteins while concurrently blocking th eir proteolysis. Compounding these deleterious effects is its promotion of satellite cell differentiation i nto myofibroblasts that produce additional collagen I. In addition, TGF has also been implicated in diseases involving fibrosis such as muscular dystrophy and inflammatory myopathy; its presence has been localized to those areas between

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23 myofibers as wel l as regions of neutrophil and macrophage infiltration. Furthermore, Smith et al. determined that TGF muscle injury (28) This is consistent with the peak activity of macrophages, suggesting a corr elation between macrophage concentration and TGF of special interest to those studying fibrotic muscle disorders, and has potential for therapeutic applications. It has also recently been postulated that nitric oxide and TGF have opposing effects. In a study investigating crush injured muscle and synovium of controls versus those of rats treated with n nitro L arginine methyl ester (L NAME), a nitric oxide synthase (NOS) inhibitor, TGF he control animals were elevated at first but subsided after three weeks. In stark contrast, the TGF NAME treated counterparts were interminably elevated, with no signs of recession even after five weeks. This relatively perpetual eleva tion of TGF in recovering muscle has been well documented to be correlated with increased collagen secretion by fibroblasts, the accretion of which results in the aforesaid deficits in muscle regeneration and tendon healing (15) Additionally, it has been shown that subjects provided with L NAME two hour s after injury have increased expression of TGF inhibitory mechanism (17) Along with the aforementioned NO hat expression, such as the collagens and proteoglycans (33) It is well within the realm of TGF arger

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24 extent, muscle recovery as a whole, is largely regulated through the NO/TGF other words, it is the incessant battle for supremacy between satellite cell activation and collagen deposition that ultimately determines the fate of the muscle. Getting to the Source of Age Related Deficits Recent research has brought Notch/Delta signaling to the forefront of the conversation when it comes to regulation of satellite cell activation. Notch has been identified as a regulator of stem cell differenti ation during embryonic development, and has a significant role in muscle regeneration. Notch signaling is initiated when one of its ligands, such as Delta, interacts with the transmembrane Notch receptor. This results in a cascade of reactions and cleavage s that culminates in the transcription of myogenic proteins via the Hey and Hes genes (Figure 1) (1) (31) Figure 2 1 Overview of Notch/Delta signaling Not only has it has been shown that Notch signaling components are upregulated in the hours to days following muscle damage, their increased activity has been

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25 localized to the satellite cells and adjacent muscle fibers themselves. This knowledge served as the impetus for further elucidation of their role, and it is now known that augmented Notch signaling promotes satellite cell proliferation while simultaneously arresting myoblast differentiation. However, when Notch signaling is inhibited by its antagonis t, Numb, proliferation is suppressed and differentiation increased. Moreover, Conboy et al. demonstrated that satellite cells in aged muscle failed to upregulate Delta expression in response to injury, significantly hampering regeneration, and more specifi cally, the proliferation of satellite cells. It is notable that increased Delta expression was correlated with decreased Numb expression. The investigators then administered Jagged Fc fusion protein, a specific inhibitor of Notch activity, to the young ani mals and similar detriments in muscle regeneratio n to that of the old tissue were observed. Furthermore, they specifically activated Notch signaling using an antibody and it elicited an almost completely identical regenerative response between the young an d old muscle (11) In a separate experiment, Conboy et al. used a heterochronic parabiosis model to determin e if exposure to a young systemic environment could rescue the regenerative capacity of old muscle. Strikingly, they demonstrated that, when exposed to young serum, aged muscle regenerated in a similar, robust fashion to that of its younger parabiont, like ly due to the measured increase in Delta expression. In contrast, the old isochronic parabionts were characterized by lackluster muscle regeneration and satellite cell activation, with notable fibrosis, a response typical of old muscle. Likewise, the young heterochronic parabionts exhibited slightly decreased upregulation of Delta, suggesting the old serum has a negative impact on the Notch pathway. In conjunction with these measurements, hepatocyte proliferation was also

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26 recorded, and the results were simi lar: increased progenitor cell proliferation in old heterochronic parabionts in relation to their old isochronic counterparts (12) Therefore, it can be postulated that the ob served age related difference in Notch activation, specifically by the ligand Delta, is the rate limiting factor present in aged serum and is responsible for its inability to activate satellite cells, thus limiting regenerative potential in aged animals re lative to their younger counterparts. Clearly, more research needs to be done to fully elucidate the mechanisms, but the therapeutic potential for the rescue of progenitor cell function is unbounded. Osteopontin (OPN) is an extracellular structural protein originally discovered in bone, which has a wide variety of functions including regulation of inflammation and wound healing. It can also function as a pleotropic cytokine, expressed primarily in macrophages, that has profound effects on muscle regenerati on as well as a myriad of other processes (21) Furthermore, Paliwal et al demonstrated that when OPN was neutralized, myogenic response increased. A lso of note was their finding that, while the quantities present in each respective muscle were equivalent, young CD11b+ macrophages exhibited a rescue effect upon muscle regeneration compared to the old macrophages when all other factors were controlled f or (27) This discovery is very intriguing especially when the Notch experiments are taken into account. It is reasonable to postulate that OPN could be one of many significant contributors to reduced Notch Delta signaling, and thus reduced regeneration, in senescent muscle. L A rginine and ISDN Exogenous NO donor supplementation is thought to increase the efficacy of the muscular regenerative process as NO plays a role in multiple stages of recovery. L arginine is the primary substrate for the synthesis of NO, acting through the nitric oxide

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27 synthase enzyme (NOS) to create NO and citrulline, which than can be converted to L arginine and begin the proce ss anew. A recent study has shown that L arginine supplementation attenuates the decline in NO content of hind limb suspended rats, as well as ameliorates the atrophic response relative to their control counterparts (20) Furthermore, an investigation by Neto et al. demonstrated that L arginine enhances muscle regeneration in an envenomation model, with treated animals showing a greater response than untreated controls but not recovering to the level of the uninjured cohort (25) The precise mechanism for these effects remains unclear, but it seems to be related to NO production by way of NOS. In addition to upregulating NO production, L arginine supplem entation has also been shown to be angiogenic. Therefore, it is not unreasonable to theorize at least some of its positive effects on muscle regeneration stem from its capacity to restore the damaged vasculature surrounding the muscle. Nevertheless, a majo r obstacle inhibiting L arginine from achieving high efficacy is its propensity to cause undesirable gastrointestinal side effects, such as nausea and diarrhea, especially in higher doses (16) Another NO donor, Isosorbide Dinitrate (ISDN), has recently entered the supplement conversation largely due to its status as a direct NO donor, rendering NOS activity irrelevant. In contrast with L arginine ISDN has very few side effects, making it an attractive alternative. In a study by Marques et al., mdx mice tibialis anterior muscle was injected with bupivacaine and subsequently supplemented with ISDN, with an additional group administered a purely vasodilatory agent The gro up given the ISDN showed a 20% increase in regeneration over both of the other groups, validating the assertion that the increase in NO is responsible for the augmented regeneration and not

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28 the vasodilatory mechanism (22) Both of these NO donors have established vasodilatory effects on the vasculature, and are becoming increasingly eminent in the field of muscle regeneration. However, future res earch focused on the actual mechanism and true efficacy of the augmented regeneration is needed to clear the ambiguity surrounding these two molecules. Conclusion To say the process of myogenesis is mediated by a myriad of factors is an egregious understat ement. From macrophages, to satellite cells, and even the recently prominent Notch signaling, it is clear we have significant work ahead of us in truly understanding the process of muscle regeneration. Nevertheless, recent research has provided the impetus for further investigation into this convoluted pathway, and its elucidation will surely prove invaluable. Furthermore, new supplementation options to improve muscle regeneration could have widespread clinical influence in the future, and the potential to rescue aged muscle with young serum is beyond promising. All of this progress makes it irrefutably evident that the means to attenuate the decline in skeletal muscle as we age exist; we just have to find them.

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29 CHAPTER 3 METHODS Experimental Design The Un iversity of Florida Institutional Animal Care and Use Committee approved the protocol of this study. Mice were exposed to a control diet, or a diet supplemented with either L arginine or isosorbide dinitrate (ISDN) for one week prior to injury to the right tibialis anterior by injection of cardiotoxin. The left tibialis anterior served as a contralateral non injured control. Mice were monitored daily for food intake and changes in body weight. At 5 and 10 days post injury, mice were killed and the tibialis anterior muscles removed for subsequent histological sectioning. The design is illustrated in Table 3 1. Table 3 1. Experimental Design 5 day Recovery 10 day Recovery Young Adult Control n=4 n= 4 Old Adult Control n= 4 n= 4 L arginine n= 4 n= 4 ISDN n= 4 n= 4 Animals Male C57 mice were purchased from the NIA aged rodent colony at 3 and 23 months of age. The animals were housed in the SPF facility in the J. Hillis Miller Health Science Center at the University of Florida and maintained on a 12:12h light dark photoperiod. Mice were housed for at least one week prior to experiments.

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30 Experimental Diets To begin the protocol, mice were randomly assigned to isocaloric low protein (10% kcal from casein) diets supplemented with either 1) L arginine (2% kcal from L arginine ), 2) a mixture of amino acids (2% kcal) specifically design to exclu de L arginine and other amino acids involved in endogenous production of L a rg inin e (Control diet), or 3) the control diet supplemented with isosorbide dinitrate (ISDN, 4 g/kg diet). Mice began experimental diets 7d prior to myotoxin injections, and continued on these diets throughout the recovery period. Experimental Protocols Cardioto xin Injury Muscle regeneration was induced by a direct transcutaneous intramuscular injection of cardiotoxin from Naja mossambica mossambica venom (Sigma Chemcial) in physiological saline, into the tibialis anterior (TA) muscles of adult mice. Injections w ere performed unilaterally, with the contralateral muscles serving as non regenerating controls. Mice were anesthetized with 5% inhaled isoflurane in oxygen, and the lower legs cleaned with 70% ethanol. A 28 gauge needle was then inserted through the skin and into the tibialis anterior muscle along the longitudinal ax is of the muscle to inject ~20L of sterile saline containing 10 M cardiotoxin. The needle was withdrawn and re inserted into the muscle multiple times at slightly d ifferent angles, injecting ~20L each time until a total of 1 00L of cardiotoxin solution had been injected. Contralateral legs were prepared identically to the injured leg, but without toxin injection. Tissue Removal and Preparation At the end of the protocol, the tibialis anterio r muscles from each leg were removed under general anesthesia (5% isoflurane) weighed, embedded in OCT

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31 (Tissue Tek) compound at resting length and frozen in liquid nitrogen cooled isopentane Samples were then cryosectioned at 10 m for histological anal yses. Following cryosectioning, muscle sections were affixed to glass microscope slides. Multiple serial sections were collected on separate slides for independent staining procedures, as described below. Hematoxylin & Eosin Stain Muscle sections were sta ined in accordance with standard hematoxylin and eosin protocols. Briefly, slides with muscle sections were air dried at room temperature for 30 min before incubation in Harris Hematoxylin solution for 5 min. After rinsing in running tap water, slides were immersed 6 times in acid alcohol (1% HCl in 70% ethanol), washed again in running tap water, and immersed briefly in ammonia water (1mL NH4OH in 1 liter of dH2O). This was followed by another rinse in water and incubation in Eosin Y solution (5% aqueous s olution) for 3 min. Slides were then rinsed again in running water, dehydrated by immersion in progressively increasing concentrations of ethanol (35, 50, 75, and 100% EtOH), and cleared by repeated immersion in CitriSolv (Fisher Scientific). Finally, drie d muscle sections were covered with permount and cover slipped. Analyses Image Analyses Microphotometric digital images of stained muscle sections were captured using a Zeiss Axiovert200 light microscope (Thornwood, NY) and Qimaging RETIGA EXi digital came ra (Surry, BC, Canada) and software (IPLab3.6.5, Scanalytics, Rockville, MD). H&E stained images were evaluated for myofiber dimensions (cross sectional area and nuclei/fiber) and myofiber density (myofiber area/total area) using ImageJ

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32 imaging software (N IH). Two images of each muscle section were captured. Within each image all fibers completely contained in the field of view were analyzed for area and nuclear content. Regeneration I ndex (RI) was tabulated through quantitative analysis of central ly situat ed nuclei divided by total number of nuclei Further, the total number of nuclei per muscle cross sectional area was determined by counting all nuclei in the field of view. Area measurements were calibrated using the captured image of a stage micrometer at the same magnification (40x) used for muscle section analysis. Statistical Analyses Myofiber cross sectional area, nuclei per fiber, regeneration index, and nuclear density per muscle area from eac h analyzed image was averaged to yield one observation pe r variable per muscle section. These data were then analyzed using a one way ANOVA followed by Tukey version 6.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com ). Significance was established at P<0.05.

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33 CHAPTER 4 RESULTS NO Augmenting Supplements as a Method of Nullifying Age Associated Muscle Wasting In this study, we investig ated the potential of two NO augmenting supplements, L arginine and i sosorbide dinitrate (ISDN) to combat the oft observed deficit in myogenic capacity of elderly individuals. Our two main experimental groups, five and ten days post CTX injection, provided two very dichotomous snapshots into the chronology of the regenerative process. Our four subgroups, you ng control (YC), old control (OC), old arginine (OA), and old ISDN (OI) allowed us not only to compare the supplemental groups with the old controls, but with the young controls as well to provide an index of restored myogenic capacity. Subject and sample metrics are shown in Table 4 1 and Table 4 2 Representative H&E ima ges of all groups are shown in F igure 4 1 Table 4 1 Avera ge tibialis a nterior ( TA ) and body m asses (BM) for all groups Values are mean SEM. 5 Day 10 Day CTX TA(mg) CL TA(mg) BM (g) CTX TA (mg) CL TA (mg) BM (g) YC 43.8 4.3 63.5 2.8 30.3 1.0 46.0 4.3 51.7 2.6 29.4 0.2 OC 46.5 1.9 60.5 1.5 37.4 1.5 39.9 3.0 47.9 0.8 35.6 0.2 OA 45.6 3.8 62.0 3.5 35.9 1.4 41.8 2.0 55.0 4.7 36.2 2.0 OI 46.8 1.4 60.8 2.5 36.0 0.9 45.1 6.3 57.1 1.6 32.2 2.1 CTX=Cardiotoxin, CL=Contralateral non injured L Arginine Supplementation Increases the Regenerative Response of Senescent Muscle at Five Days Post Injury Our five day data illustrate that L arginine supplementation had a significant effect on muscle regeneration. The Regeneration Index (RI), calculated as centrally located nuclei per total nuclei, provides a gross but nonetheless powerful assessment of the balance between cell proliferation (including both myogenic and infiltrating immune

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34 cells) and myogenic cell fusion. The RI of the OA group was significantly different from that of the OC group, and was nearly equal to that of the YC group (Figure 4 3A). However, the average cross sectional area and the total fiber area showed no change (Figure 4 2A; Figure 4 4A). In addition, the response at ten days was equally insignificant across all categories (Figure 4 2B; Figure 4 3B; Figure 4 4B). Also of note, th e average cross sectional area of the cells in the OA group was, in general, less than those of the other groups. This is due to the fact that, on average, the OA samples were characterized by more abundant, but smaller cells, than those of the other group s, as noted during observation and data collection. Table 4 2. Recorded and calculated parameters of regeneration for all samples TA CXA F ibers N uclei RI Cen tral Nuc lei Tot al Area Five days post CTX injection YC 963 77 42.3 2.4 490 25 0.194 0.007 95.1 4.3 40134 3382 OC 624 46 52.8 7.0 608 11 0.143 0.162 87.1 11.4 32100 3382 OA 638 54 52.6 4.1 586 22 0.198 0.004 116.0 6.1 32919 456 OI 668 63 40.5 3.2 531 13 0.178 0.015 94.4 6.5 28262 4412 Ten days post CTX injection YC 1997 258 49.8 6.5 481 30 0.289 0.013 128 11.1 94417 4337 OC 1289 164 66.4 8.4 465 47 0.263 0.014 119 8.5 84843 12029 OA 978 107 93.8 10.2 549 47 0.250 0.026 135 11.3 89406 7658 OI 1314 124 82.6 7.8 574 41 0.243 0.017 148 11.9 103229 4558 Values are meanSEM. CXA=Cross sectional area, RI= Regeneration Index

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35 Figure 4 1 Representative H&E sections from all groups. TA muscle was removed at 5 or 10 days and analyzed using ImageJ. 5D CTX 5D CTX 10D CTX

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36 Figure 4 2 Average TA cross sectional area post CTX injection. *Significantly different from YC (p<0.05) A) Five da ys post CTX injection B) Ten days post CTX injection A B

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37 Figure 4 3 Regeneration Index (RI) of all groups post CTX injection. # Significantly different from YC (p<0.05). *Significantly different from OC (p<0.05) A) Five days post CTX injection. B) Ten days post CTX injection A B

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38 Figure 4 4 To tal fiber area for all group s post CTX injection A) Five days post CTX injection. B) Ten days post CTX injection A B ISDN Supplementation Shows a Positive Trend Toward Increasing the Myogenic Capacity of Senescent Muscle Treatment with ISDN showed a positive, yet not statistically significant response on the RI of the subjects at five days post CTX injection (Figure 4 3A). However, similar to the arginine treatment group, the ISDN supplementati on at ten days seemed to have

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39 negligib le effect s across the board (Figure 4 2B; Figure 4 3B; Figure 4 4B). ISDN also seemed to have a slightly more positive effect than L arginine on TA cross sectional area at five days even though the total fiber area was much lower, possibly due to the relat ively large disparit y in fiber number (Figure 4 4A; Table 4 2 ).

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40 CHAPTER 5 DISCUSSION Principal Findings The impetus for this study was provided by prior forays into the investigation of in vitro L arginine and ISDN supplementation, and the resulting increase in NO levels, as a means of combating the oft reported phenotypic inability of aging muscle to regenerate in any sort of accordance with that of its younger counterparts. In 2008, Betters et al. reported an incr ease in satellite cell activation as a result of L arginine supplementation using a cell culture mode l as well as a full rescue response when HGF was administered. However, the results obtained with L arginine failed to demonstrate a recovery to youthful levels, but reported a nonetheless important partial vitiation of the deficit. They then postulated that the difference in the regenerative capacity of aging muscle fibers was largely a construct of the decreased sensitivity of the older satellite cells to NO, and not the inherent quantitative presence of NO itself although the augmented presence can have limited positive effects (4) This work, in addition to that conducted by Marques et al. demonstrating the potential for ISDN to improve myogenesis in an mdx model, largely provided the foundation for the current study to investigate the ability of NO donor supplementation to at least partially mitigate the a forementioned myogenic deficit. This study is the first to demonstrate the potential for exogenous L arginine and ISDN supplementation to improve senescent muscle regeneration at five days post muscle injury. Our results, despite some of them lacking proper stat istical significance, dis play a strong trend toward the chosen supplements facilitating muscle regeneration. As such, these results tentatively confirm our prior hypotheses.

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41 M atrix Metalloproteinases are Key Modulators of Satellite Cell Function The deficit in aging satellite ce ll function has been well documented, and recent evidence has implicated the aging environment acting to retard the activation of the satellite cell, as the culprit, rather than the satellite cell itself. Rando and colleagues have illustrated on multiple occasions that this is the case, often using young serum to rescue the regenerative potential of the aged muscle and produce levels of satellite cell activation akin to the levels achieved in youth (12) In addition, a s noted by Yamada et. al, a family of ubiquitous endopeptidases known as matrix metalloproteinases (MMPs) especially MMP 2, have the ability to cleave hepatic growth factor (HGF) from its extracellular bind, freeing it to activate satellite cells (33) Moreover, it has been shown by multiple studies that MMP dysreg ulation is a common problem associated with age and is involved in the formation of many serious diseases, such as cancer and cardiovascular disease (3) (10) (19) (32) Combined with the knowledge that senescent satellite cell activation is compl etely rescued through HGF supplementation and the fact that proper MMP activation is NO dependent, it would follow that the primary mechanism through which regeneration is increased in elderly subjects could very well be the corresponding rise in proper ac tivity of the MMPs achieved through NO supplementation and the resulting increases in bioavailable HGF. Therefore, the data suggests the relatively robust regenerative capacities exhibited by the treatment groups in our data at five days are a t least parti ally a result of improved MMP signaling through facilitated NO production. NO Supplementaion as a Means of Sarcopenia Treatment Despite the rampant prevalence of sarcopenia in modern society, there has been a relative lack of research into possible treatm ents and therapeutic methods. A few

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42 studies, however, have been promising using mdx models of sarcopenia. This clinical model may be considered a form of accelerated aging since mdx mice lack sufficient levels of nNOS, the principal source of muscular NO and exhibit severe deficits in muscle regenerative potential at a relatively young age A stage I clinical trial conducted ibuprofen may be effective in treating sarcopenia. Many of the patients included in the trial not only had improved motor function, but also had decre a sed serum levels of TGF (14) As mentioned earlier, increased TGF satellite cell activation and an op posing increase in collagen deposition and fibrosis in the muscle, common characteristics of senescent myofibers. It is therefore not unreasonable to hypothesize that the positive results of ISDN in this study could be due to the increased levels of NO res ulting in enhanced MMP activation and signaling. Combined with our data, this suggests that supplementation with ISDN could be an effective method in the prevention and attenuation of the muscle wasting that occurs in sarcopenia. Furthermore, a study by Ti dball et al. has shown that magnified NO production effectively inactivates the proteolytic actions of calpain, thought to be a prominent player in sarcopenia. They postulated that through the increased activity of nNOS, calpains were S nitrosylated, signi ficantly attenuating the loss of muscle tissue usually associated with aging. Taken collectively, these studies along with our data indicate that both ISDN and L arginine supplements could be potential ly efficacious therapeutic interventions in the treatme nt of sarcopenia. Possible Side Effects of L A rginine Despite its clear budding promise as a solution to the rampant issue of sarcopenia, it would be a critical omission to not discuss the potential side effects of L

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43 arginine supplementation. As previously mentioned, common side effects reported by those administered L arginine include nausea and diarrhea, as well as numerous other symptoms associated with gastrointestinal distress (16) However, there has been a relatively recent revelation that prolonged L arginine supplementation results in increased fibrosis. Tidball et al. recently conducted a study investigating the effects of long term L arginine supplementation in mdx subjects on muscle fibrosis, and the results were striking: fibrosis increased markedly both in the skeletal muscles as well as the myocardium. This occurred as a result of the upregulati on of arginase, an enzyme prevalent in the metabolism of arginine and its effect on macrophages, causing fibrosis of the muscle tissue (32). It is important to not e, however, the facts that mdx subjects are by nature characterized by a dearth of intrinsic NOS and that NOS is a primary competitor of arginase. Therefore, caution should be demonstrated before extrapolating these results to healthy individuals, because the lack of dystrophin at the membrane could very well be a confounding factor, and much more research needs to be done in this area to assure correct applic ation. Overall, the future of L arginine supplementation as a potential option in the treatment of senescence related muscle wasting is very bright, but one that is wrought with questions and we suspect many more years of inquiries into its far reaching ef fects will be required to elucidate its full potential. Future Directions When the current literature is taken collectively into account with our data, it is clear that there is much work to be done. First, any future studies in our lab concerning this ma tter would need to include contractile data to ascertain the effects NO donor supplementation has, if any, on functional recovery. Second, many more time points are needed to address the different stages of muscle recovery and the potential impacts

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44 supplem ents are having at the given points. Our results show a clear effect at five days, but the effect is nearly nonexistent at ten days. Repeated studies in this area would help to solidify the knowledge base on this matter, and paint a clearer picture of the cellular events between those two time points. Further as with any animal model, extrapolating these results to humans is wrought with hazards and human research would be the final step in verifying the efficacy of such supplements in humans. Conclusions Based on our data and the body of work currently presented in the literature, we believe that ISDN and L arginine supplementation as a means of attenuating the effects of sarcopenia definitively merits further investigation. While not without its seemingl y infinite facets, the culprits responsible for the myogenic deficit in the elderly are slowly coming into view. We postulate that nNOS and MMPs are dysregulated in the elderly account for a significant portion of the regenerative dearth observed in sarco penia, and that L arginine and ISDN supplementation have the potential to partially mitigate that deficit through the promotion of nNOS presence and proper MMP function.

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45 LIST OF REFERENCES 1. Artavanis T sakonas S, Rand MD, Lake RJ Notch Signaling: Cell Fate Control and Signal Integration in Development. Science 284: 770 776, 1999. 2. Asakura A, Rudnicki MA, Komaki M Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68: 245 253, 2001. 3. Barani AE, Durieux A C, Sabido O, Freyssenet D Age related changes in the mitotic and metabolic characteristics of muscle derived cells. J. Appl. Physiol. 95: 2089 2098, 2003. 4. Bet ters JL, Lira VA, Soltow QA, Drenning JA, Criswell DS Supplemental nitric oxide augments satellite cell activity on cultured myofibers from aged mice. Experimental Gerontology 43: 1094 1101, 2008. 5. Bogdan C Nitric oxide and the regulation of gene expr ession. Trends in Cell Biology 11: 66 75, 2001. 6. Brack A, Rando T Intrinsic Changes and Extrinsic Influences of Myogenic Stem Cell Function During Aging. Stem Cell Reviews and Reports 3: 226 237, 2007. 7. Butterfield TA, Best TM, Merrick MA The Dual Roles of Neutrophils and Macrophages in Inflammation: A Critical Balance Between Tissue Damage and Repair. Journal of Athletic Training 41: 457, 2006. 8. Cantini M, Carraro U Macrophage released Factor Stimulates Selectively Myogenic Cells in Primary Mus cle Culture. Journal of Neuropathology 54: 121 128, 1995. 9. Charg SBP, Rudnicki MA Cellular and Molecular Regulation of Muscle Regeneration. Physiol Rev 84: 209 238, 2004. 10. Chiao YA, Ramirez TA, Zamilpa R, Okoronkwo SM, Dai Q, Zhang J, Jin Y F, Lin dsey ML Matrix metalloproteinase 9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice. Cardiovasc. Res. 96: 444 455, 2012. 11. Conboy IM, Conboy MJ, Smythe GM, Rando TA Notch Mediated Restoration of Regenerative Potential t o Aged Muscle. Science 302: 1575 1577, 2003. 12. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433: 760 764, 2005. 13. Conboy IM, Rando TA Hete rochronic parabiosis for the study of the effects of aging on stem cells and their niches. Cell Cycle 11: 2260 2267, 2012.

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46 14. Comi GP, Turconi AC, Magri F, Stefanoni G, Brunelli S, Bresolin N, Cattaneo D, Clementi E Nitric oxide donor and non steroidal anti inflammatory drugs as a therapy for muscular dystrophies: Evidence from a safety study with pilot efficacy measures in adult dystrophic patients. Pharmacological Research 65: 47 2 479, 2012. 15. Darmani H, Crossan J, Mclellan SD, Meek D, Curtis A Expression of nitric oxide synthase and transforming growth factor beta in crush injured tendon and synovium. Mediators of Inflammation 13: 299 305, 2004. 16. Evans RW, Fernstrom JD, T hompson J, Morris Jr SM, Kuller LH Biochemical responses of healthy subjects during dietary supplementation with L arginine. The Journal of Nutritional Biochemistry 15: 534 539, 2004. 17. Filippin LI, Moreira AJ, Marroni NP, Xavier RM Nitric oxide and r epair of skeletal muscle injury. Nitric Oxide 21: 157 163, 2009. 18. Gros J, Manceau M, Thom V, Marcelle C A common somitic origin for embryonic muscle progenitors and satellite cells. Nature 435: 954 958, 2005. 19. Jiang L, Zhang J, Monticone RE, Tell johann R, Wu J, Wang M, Lakatta EG Calpain 1 regulation of matrix metalloproteinase 2 activity in vascular smooth muscle cells facilitates age associated aortic wall calcification and fibrosis. Hypertension 60: 1192 1199, 2012. 20. Lomonosova Y, Kalamkar ov G, Bugrova A, Shevchenko T, Kartashkina N, Lysenko E, Shvets V, Nemirovskaya T Protective effect of L arginine administration on proteins of unloaded <i>m. soleus</i> Biochemistry (Moscow) 76: 571 580, 2011. 21. Lund SA, Giachelli CM, Scat ena M The role of osteopontin in inflammatory processes. Journal of Cell Communication and Signaling 3: 311 322, 2009. 22. Marques MJ, Luz MAM, Minatel E, Neto HS Muscle regeneration in dystrophic mdx mice is enhanced by isosorbide dinitrate. Neuroscien ce Letters 382: 342 345, 2005. 23. McClung JM, Davis JM, Carson JA Ovarian hormone status and skeletal muscle inflammation during recovery from disuse in rats. Exp Physiol 92: 219 232, 2007. 24. Morris SM Arginine: beyond protein. Am J Clin Nutr 83: 508S 512S, 2006. 25. Neto HS, Vomero VU, Marques MJ l arginine enhances muscle regeneration after experimental envenomation by B. jararacussu: A future for nitric oxide based therapy? Toxicon 48: 353 357, 2006.

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47 26. Ono Y, Boldrin L, Knopp P, Morgan JE, Zammit PS Muscle satellite cells are a functionally heterogeneous population in both somite derived and branchiomeric muscles. Developmental Biology 337: 29 41, 2010. 27. Paliwal P, Pishesha N, Wijaya D, Conboy IM Age dependent increase in the leve ls of osteopontin inhibits skeletal muscle regeneration. Aging (Albany NY) 4: 553, 2012. 28. Smith CA, Stauber F, Waters C, Alway SE, Stauber WT Transforming growth factor J Appl Physiol 102: 755 761, 20 07. 29. Sorichter S, Mair J, Koller A, Gebert W, Rama D, Calzolari C, Artner Dworzak E, Puschendorf B Skeletal troponin I as a marker of exercise induced muscle damage. J Appl Physiol 83: 1076 1082, 1997. 30. Sorichter S, Mair J, Koller A, Muller E, Kre mser C, Judmaier W, Haid C, Calzolari C, Puschendorf B Creatine kinase, myosin heavy chains and magnetic resonance imaging after eccentric exercise. Journal of Sports Sciences 19: 687 691, 2001. 31. Tsivitse S Notch and Wnt Signaling, Physiological Stim uli and Postnatal Myogenesis. International Journal of Biological Sciences (2010). doi: 10.7150/ijbs.6.268. 32. Wang G, Wang W, Zhou J, Yang X Correlation between telomerase activity and matrix metalloproteinases 2 expression in gastric cancer. Cancer Bi omark 13: 21 28, 2013. 33. Yamada M, Sankoda Y, Tatsumi R, Mizunoya W, Ikeuchi Y, Sunagawa K, Allen RE Matrix metalloproteinase 2 mediates stretch induced activation of skeletal muscle satellite cells in a nitric oxide dependent manner. The International Journal of Biochemistry & Cell Biology 40: 2183 2191, 2008.

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48 BIOGRAPHICAL SKETCH Kevin James Miller was born in 1988 in Dunedin, Florida to Joseph and Patricia Miller. He attended and graduated from Olympia High School in 2007 and begun attending the University of Florida th e following in the same subject under the supervision of Dr. David Criswell He has also worked for UF recreational sports for 5 of his 6 years at the university, and has been a certified personal trainer since 2011. He was recognized as the 2011 2012 UF personal trainer of the year in April 2012, and was afforded the opportunity to present a lecture at the 2013 Evolve fitness symposium. Following graduation, he will be employed at the Equinox fitness club in Coral Gables, Florida, as a personal trainer, pursuing his dream of helping any and ever yone fully realize and redefine their physical potentials.