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Nitric Oxide Synthase Activity Affects Gene Expression in Overloaded Skeletal Muscle

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PAGE 1

NITRIC OXIDE SYNTHASE ACTIVITY AFFECTS GENE EXPRESSION IN OVERLOADED SKELETAL MUSCLE By JEFF E. SELLMAN 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 2005

PAGE 2

Copyright 2005 by Jeff E. Sellman

PAGE 3

To my parents, Richard and Rebecca Sellman, for their unconditional love and limitless support

PAGE 4

ACKNOWLEDGMENTS First and foremost, I would like to thank my parents (Rich and Rebecca Sellman) for their unconditional and continued support. They have been both my life preserver and my kayak when I needed help in keeping my head above water. Without their love, I would run the risk of losing sight of the bigger picture. This project was a part of Dr. David Criswells vision of his fledgling laboratory. As my committee chair and mentor, Dr. Criswell showed limitless patience, direction, and faith in entrusting me with this project. I thank him for supplying me with the tools (both tangible and not) to see the project to completion. I acknowledge all of the graduate students and professors in the Center for Exercise Science who witnessed my progression from an undergraduate student to a graduate assistant. I especially thank Keith DeRuisseau for his knowledge of and input into my results. Finally, I thank Dr. R. Andrew Shanely and the Frank Booth laboratory for technical assistance in running the assays. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Problem Statement........................................................................................................2 Hypotheses....................................................................................................................3 Definition of Terms......................................................................................................3 Limitations/Delimitations/Assumptions.......................................................................6 Significance of the Study..............................................................................................7 2 LITERATURE REVIEW.............................................................................................8 Skeletal Muscle Adaptations to Load...........................................................................8 Hypertrophy and Fiber Type Shifts with Loading........................................................9 Skeletal Muscle Hypertrophy and Insulin-Like Growth Factor (IGF-1) Signaling...10 Skeletal Muscle Hypertrophy and Vascular Endothelial Growth Factor (VEGF).....14 Hepatocyte Growth Factor (HGF) and Muscle Growth.............................................16 Myogenin (MGN) and Control of Adult Muscle Phenotype......................................18 Hypertrophy and Nitric Oxide....................................................................................19 Summary.....................................................................................................................21 3 METHODS.................................................................................................................23 Animals.......................................................................................................................23 Synergist Ablation Surgery.........................................................................................24 Experimental Protocol................................................................................................25 Nitric Oxide Production..............................................................................................26 Reverse Transcription and Real-Time Quantitative PCR...........................................26 Semi-Quantitiave RT-PCR.........................................................................................27 v

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Immunoblotting..........................................................................................................28 Limitations..................................................................................................................29 Vertebrate Animals.....................................................................................................30 Statistical Analysis......................................................................................................30 4 RESULTS...................................................................................................................31 Systemic and Biological Responses to Treatment......................................................31 Myogenin mRNA Expression.....................................................................................31 Contractile Protein mRNA Expression.......................................................................32 Growth Factor mRNA Expression..............................................................................32 Phosphorylation of p70 S6K ..........................................................................................33 iNOS Protein Expression............................................................................................33 5 DISCUSSION.............................................................................................................40 Skeletal -Actin mRNA Expression..........................................................................41 VEGF Expression.......................................................................................................42 IGF-1 Expression and Phosphorylation of p70 s6 Kinase............................................42 Future Directions........................................................................................................43 Conclusions.................................................................................................................45 LIST OF REFERENCES...................................................................................................46 BIOGRAPHICAL SKETCH.............................................................................................54 vi

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LIST OF TABLES Table page 4-1 Body mass, plantaris mass, and total protein data for the overloaded rats...............33 4-2 Real-time PCR quantification of mRNA transcripts for selected growth factors and a regulatory gene in the plantaris muscle..........................................................34 vii

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LIST OF FIGURES Figure page 3-1 Experimental design flowchart.................................................................................23 4-1 Real Time PCR assessment and quantification of contractile protein mRNA transcripts.................................................................................................................35 4-2 Real Time PCR quantification of insulin-like growth factor mRNA transcripts.....36 4-3 Semi-quantitative RT-PCR analysis of VEGF mRNA splice variant expression....37 4-4 Western blot analysis of p70 s6K ................................................................................38 4-5 Immunoblot assessment of iNOS protein expression.............................................39 viii

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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 NITRIC OXIDE SYNTHASE ACTIVITY AFFECTS GENE EXPRESSION IN OVERLOADED SKELETAL MUSCLE By Jeff E. Sellman December 2005 Chair: David Criswell Major Department: Applied Physiology and Kinesiology Nitric oxide is a mechanically sensitive signal in skeletal muscle. Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth after 5 days of functional loading. We also tested the hypothesis that NOS activity is necessary for functional overload-induced upregulation of growth factors, myogenin, and contractile gene mRNAs in the rat plantaris muscle. Twenty-four female Sprague-Dawley rats (~250 g) were randomly divided into three groups (n=8/group): Control, N-nitro-L-arginine methyl ester (L-NAME: 100 mg/kg/d), or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM: 10 mg/kg/d). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris for 5 days. Sham surgery was performed on the left hindlimb, which served as a normally loaded (NL) control. No group differences were observed among NL muscles. Real-time PCR analyses showed elevated (p<0.05) mRNA expression for insulin-like growth factor-1 (IGF-1), mechanoix

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growth factor (MGF: load-sensitive splice variant of IGF-1), hepatocyte growth factor (HGF), and myogenin and reduced (p<0.05) total VEGF mRNA expression in the OL muscle compared to NL. Neither L-NAME nor TRIM affected HGF, VEGF, or myogenin responses. However, OL-induction of IGF-1 and MGF mRNA was greater (P<0.05) in the TRIM group compared to the Controls. Conversely, overload-induction of phosphorylated p70 S6 kinase (p70 s6K ) was prevented in the TRIM group. Type I (slow) myosin heavy chain (MHC) and skeletal -actin mRNAs were increased in the Control/OL muscle (an effect that was completely prevented in both NOS-inhibitor groups). Therefore, nNOS activity is necessary for overload-induction of Type I (slow) MHC and skeletal -actin mRNA and p70 S6K phosphorylation. Further, the inhibition of nNOS causes a compensatory increase in IGF-1 expression during overload. x

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CHAPTER 1 INTRODUCTION Skeletal muscle is an extremely plastic tissue. Variations in external load result in structural, biochemical, and morphological adaptations in muscle fibers. Specifically, alterations in mitochondrial number, enzymatic profile, structural protein expression and content, and capillary angiogenesis accompany both muscle atrophy and hypertrophy. Chronic overload causes dramatic muscle growth. This hypertrophy response involves the activation and later fusion of satellite cells to the muscle fibers (57, 58), and increased synthesis of structural and contractile proteins (28, 76). Concurrently, capillary angiogenesis is induced to support growing muscle (54). The increased fiber recruitment associated with muscle overload also causes an up-regulation of slow fiber type-specific genes. Molecular regulation of growth factors and transcription factors that govern muscle growth is poorly understood. Therefore, discovering the molecules responsible for signaling this coordinated response is vital for understanding load-induced adaptive changes in skeletal muscle. Background Loss of skeletal muscle mass is a serious clinical problem in disease states (such as cancer and AIDS) and in conditions such as prolonged bed rest and spaceflight. Preserving muscle mass by attenuating muscle loss and/or stimulating muscle growth can be vital to decreasing recovery time and increasing the patients ability to ambulate freely and enjoy independence. Muscle growth in response to increased load is a complex event that requires the transcription of many different factors to contribute to the changing 1

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2 phenotype; therefore, it is important to understand the underlying mechanisms responsible for the transduction of mechanical forces into chemical signals. Nitric oxide (NO) is an important signaling molecule, playing a role in various physiologic processes. These effects are broad and far-reaching, as NO has been implicated in preserving endothelial function and may be necessary for the slow fiber-type transition associated with overload-induced hypertrophy. A family of nitric oxide synthases (NOS), all of which can be expressed in adult skeletal muscle, governs its synthesis. Neuronal NOS (nNOS) is the most abundant isoform in skeletal muscle, being associated with the dystrophin complex (39, 71). The activity of this enzyme is induced by contractile activity (70). Problem Statement Increased muscle load results in increased levels of growth/regulatory factors and contractile proteins, and greater nNOS activity. Therefore, we postulate that the events are related. We suggest that NO acts to coordinate the events associated with muscle growth. We tested the effects of NOS inhibition on short-term responses to chronic muscle overload. Variables in Study Independent variables. We manipulated load to the plantaris and NOS inhibition by administering N-nitro-L-arginine methyl ester (L-NAME) and 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM). Dependent variables. We measured expression of growth factors (insulin-like growth factor-1 (IGF-1), mechanogrowth factor (MGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF)); a myogenic regulatory factor,

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3 myogenin (MGN); and contractile proteins, skeletal -actin and Type I (slow) myosin heavy chain (MHC) Control variables. Gender was excluded from the study: we used only female Sprague-Dawley rats. Aging effects were also excluded: we restricted observations to young adult rats (~4 months old). Extraneous variable. We did not control for differences in the activities of the animals. Since some animals may ambulate more freely than others, the load frequency may differ among animals. However, preliminary experiments show a robust and reproducible hypertrophy of the plantaris in response to unilateral ablation of its synergists. Acute IP injections have the potential to induce a stress response that then results in a cascade of proteins that could confound the study. To control for this, each animal in the study was given an IP injection, either of TRIM or vehicle. Hypotheses 1. Five days of functional overload will increase the local expression of growth/regulatory factor mRNAs (IGF-1, MGF, HGF, VEGF, and MGN) and the local expression of skeletal -actin and Type I (slow) MHC. 2. NOS inhibition will prevent increased local expression of the growth/regulatory factors, skeletal -actin, and Type I (slow) MHC. 3. Specific nNOS inhibition will prevent increased local expression of growth/regulatory factors, skeletal -actin, and Type I (slow) MHC to the same extent as non-isoform specific systemic NOS inhibition. Definition of Terms 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM): a selective inhibitor of the neuronal isoform of NOS (nNOS). Hepatocyte growth factor (HGF): an autocrine/paracrine factor that binds to the c-met receptor. In skeletal muscle, HGF coordinates the activation and proliferation of

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4 satellite cells during muscle regeneration and growth. This factor has been identified as the component of crushed muscle extract that is capable of activating quiescent satellite cells in vitro and in vivo (67). Hypoxanthine guanine phosphoribosyl transferase (HPRT): an enzyme that plays a role in purine biosynthesis. HPRT was chosen in this model as a normalizing gene in Real-Time PCR because of its constant expression with our manipulations (overload and NOS inhibition). Insulin-like growth factor-1 (IGF-1): produced by the liver and skeletal muscle, this growth factor is an important autocrine/paracrine factor capable of stimulating proliferation and differentiation of activated satellite cells. IGF-1 also activates protein synthesis in skeletal muscle fibers via Akt/mTOR activation of protein translation. Mechanosensitive growth factor (MGF): a splice variant of IGF-1 that is particularly sensitive to load and injury in skeletal muscle. Myogenin (MGN): a myogenic regulatory factor that directs muscle differentiation. MGN expression in adult muscle fibers plays a role in directing the slow-oxidative phenotype. N G -nitro-L-arginine methyl ester (L-NAME): a non-isoform selective competitive inhibitor of nitric oxide synthase activity. Nitric oxide (NO): rapidly diffusing signaling molecule synthesized by a family of isozymes, the nitric oxide synthases. Its responses are largely mediated by S-nitrosylation of cysteine residues or by interactions with iron and copper. It has been implicated in many skeletal muscle processes, including gene expression.

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5 Nitric oxide synthase (NOS): a complex enzyme responsible for the production of NO. It acts on molecular oxygen, arginine, and NADPH to produce NO, citrulline, and NADP + The process requires five cofactors (FAD, FMN, Heme, calmodulin, and tetrahydrobiopterin) and two divalent cations (calcium and heme iron). Three distinct forms of NOS have been identified: nNOS, eNOS, and iNOS. All NOS isoforms are expressed in mammalian skeletal muscle. Normal load (NL): the contralateral (left) plantaris serves as an internal control since the gastrocnemius, plantaris, and soleus remain intact. It is assumed that the work done by the NL plantaris mimics the normal, physiologic condition. Overload (OL): the right plantaris serves as the unit that receives a higher external load due to removal of the muscles synergistic to it, the gastrocnemius and the soleus. Polymerase chain reaction (PCR): a technique that creates copies of specific DNA fragments by supplying gene-specific primers, a nucleotide pool, and DNA Taq polymerase. Reverse transcription (RT): a process that synthesizes DNA from an RNA template (isolated total cellular RNA) using the enzyme reverse transcriptase as a catalyst. Satellite cells: normally quiescent, satellite cells are committed stem cells of adult skeletal muscle. Residing between the basal lamina and the sarcolemmal surface of adult muscle fibers, they are activated by conditions such as increased load and injury. They help repair skeletal muscle and mediate muscle growth by differentiating into myocytes. Vascular endothelial growth factor (VEGF): a regulator of blood vessel synthesis induced by IGF-1 during muscle growth.

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6 Limitations/Delimitations/Assumptions Limitations. The invasive nature of this research negates the use of humans as subjects. A rat model was chosen due to genetic homogeneity that allows the researcher to draw conclusions about a population based on the sample studied. Also, rat skeletal muscle shows similarities in phenotype and genetic makeup to human muscle. Surgically induced overload provides a dramatic response in determining changes in gene expression and alterations in protein synthesis. Therefore, this model provides ease with which the researcher is able to uncover the signaling pathways responsible for the changes associated with hypertrophy. However, unilateral removal of synergists may not guarantee the animal utilizes each leg uniformly during post-surgical ambulation. Therefore, it is possible that the contralateral limb serving as the NL control may experience a small hypertrophic response should the animal favor that leg. To control for this, we performed a sham surgery to the left leg by isolating the synergists without transection. Therefore, the animal would perceive injury to both legs that would allow the ambulation to better mimic the normal, physiologic condition. Similar to previous studies using L-NAME administered in the animals drinking water, our animals showed an initial decrease in the water consumed. However, after an adjustment period, the L-NAME animals did not consume liquid at a different rate than either the TRIM or the Control groups. Finally, TRIM administered intraperitoneally (IP) may have induced an undesired stress response in the animal. Therefore, the L-NAME and Control animals received IP injection of phosphate-buffered saline (PBS) vehicle. TRIM inhibits both nNOS and, to a lesser extent, iNOS. Therefore, to assess the possible potentiation effects of iNOS

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7 inhibiton, we ran Western blots on NO and OL control plantaris to measure iNOS protein levels. Delimitations. Gender and species differences may exist in the hypertrophic response to the functional overload model. We have chosen to study female Sprague-Dawley rats. Assumptions. Since the animals were housed in pairs, water consumption was assumed to be equal between cage mates. It is also assumed that the animal did not favor the contralateral limb during the OL period. Our lab previously showed that functional overload induced by synergist ablation was a sufficient stimulus to show significant differences between the overloaded plantaris and the contralateral control. Significance of the Study Attenuating muscle wasting is a pertinent clinical goal in combating pathologies and conditions that lead to loss of function and impairment of ambulation. Aging and disease lead to impaired skeletal muscle regrowth and regeneration, resulting in loss of muscle mass and strength. Recovery from neurodegenerative disorders, spinal cord lesions, and muscle unloading due to spaceflight, injury, or extended bed rest will be more rapid if therapeutic strategies are discovered and implemented. Therefore, it is critical to determine the signaling mechanisms responsible for the load-induced phenotypic adaptations in skeletal muscle. Elucidating the biochemical pathways controlling muscle growth and the phenotypic changes that support recovery from injurious assault can lead to therapeutic strategies that prevent or possibly reverse muscle atrophy. Progression in this basic science may aid in identifying targets for anti-atrophy drugs.

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CHAPTER 2 LITERATURE REVIEW Adult skeletal muscle is highly responsive to changes in functional demands and, therefore, serves as an excellent model in elucidating the molecules that serve as links between mechanical stimuli and gene expression. Researchers employ clinically relevant in vivo models as well as in vitro models due to the ease with which the mechanical effects can be controlled and isolated. In whole animals, chronic overload causes structural and phenotypic alterations in muscle fibers, resulting in dramatic muscle growth. Likewise, myotubes subjected to stretch experience growth, proliferation and associated fiber type changes in culture. The pathways governing these changes, however, are poorly understood. Nitric oxide synthase (NOS) activity may be essential during increased contractile activity, suggesting nitric oxide (NO) may act as a coordinating master signal during muscle growth. The purpose of this study was to determine if NOS activity inhibition would attenuate the increased expression of muscle growth/regulatory factors and proteins associated with the contractile apparatus during overload-induced hypertrophy. This chapter provides a critical review of the scientific literature related to the proposed project along with interpretations where possible. Skeletal Muscle Adaptations to Load Adult skeletal muscle is extremely adaptable to changes in mechanical loading. The adaptations to chronic overload involve many coordinated steps that lead to structural and phenotypic changes within the muscle fibers. The resulting growth involves the activation and fusion of satellite cells, the increased synthesis of structural 8

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9 and contractile proteins, and subsequent capillary angiogenesis. Each of these steps is controlled by specific growth and/or regulatory factors. Hepatocyte growth factor (HGF) initiates satellite cell activation, insulin-like growth factor-1 (IGF-1) governs cell proliferation and fusion, and activates protein synthesis, vascular endothelial growth factor (VEGF) regulates angiogenesis, and myogenin (MGN) is involved in differentiation and expression of the slow phenotype. Each of these factors and its involvement in skeletal muscle growth following bouts of overload and/or stretch is described below. Hypertrophy and Fiber Type Shifts with Loading Growth of skeletal muscle fibers in response to an increased mechanical load is a complex event marked by large-scale remodeling of fiber architecture. The process is associated with injury, immune cell infiltration, and regeneration (69), and involves the activation and subsequent fusion of satellite cells to the muscle fibers (48, 57, 58), and increased synthesis of structural and contractile proteins (28, 76). Increased protein synthesis causes the accumulation of contractile and structural proteins resulting in the addition of myofibrils and an accompanying increase in fiber cross-sectional area. The increase in fiber cross-sectional area reflects an increase in muscle fiber diameter without an increase in the number of muscle fibers (26, 72). Finally, the fiber type changes associated with increased muscle recruitment are mediated via calcium-dependent signaling, including calcium-calmodulin-dependent protein kinase and calcineurin. These pathways interact with others to transcriptionally regulate expression of the myogenic regulatory factor, myogenin (77). Myogenin, in turn, plays a major role in activation of the myogenic program in differentiating satellite cells, as well as regulating the slow-oxidative phenotype in adult myofibers (34).

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10 Skeletal Muscle Hypertrophy and Insulin-Like Growth Factor (IGF-1) Signaling IGF-1 signaling cascade. IGF-1 is an important regulator molecule in muscle physiology, particularly important for maintenance and growth of adult skeletal muscle mass (3). Although the major part of the circulating fraction of total IGF-1 is produced in the liver, IGF-1 is also locally expressed in the muscle (1). This endogenous IGF-1 acts in a paracrine/autocrine manner by binding to skeletal muscle IGF-1 receptor (IGFR). IGF-1 binding to IGFR, leads to activation of the insulin receptor tyrosine kinase and subsequent activation of several cytosolic substrates, such as calcineurin, the mitogen-activated protein kinase/extracellular signal-related protein kinase (MAPK/ERK) and the phosphoinositide 3-kinase (PI3K). Interestingly, these pathways have distinctive and possibly opposing end results. Calcineurin is a phosphatase that targets a family of transcription factors, the nuclear factors of activated T-cells (NFAT). Once dephosphorylated, cytosolic NFAT translocates to the nucleus to increase the transcription of IGF-1-sensitive genes, specifically Type I (slow) myosin heavy chain (MHC). Interestingly, IGF-1 also activates MAPK/ERK, a powerful myoblast proliferative cascade that inhibits differentiation, and PI3K, whose activity ultimately leads to increased protein synthesis and differentiation. However, IGF-1 is a more powerful and potent activator of PI3K. Once activated, PI3K phosphorylates Akt/PKB and initiates a protein kinase cascade that includes mammalian target of rapamycin (mTOR), p70s6 kinase and increased protein synthesis. As a result, IGF-1 plays a major role in the growth and maintenance of adult skeletal muscle and is a potent stimulator of anabolic, mitogenic, and myogenic processes in skeletal muscle (51, 74). The IGF-1 response in functionally overloaded skeletal muscle. While circulating IGF-1 is important to these skeletal muscle responses, it is clear that the

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11 autocrine/paracrine actions of endogenously produced IGF-1 are critical for muscle hypertrophy (1). Perhaps the most convincing evidence that IGF-1 mediates adult skeletal muscle hypertrophy comes from muscle-specific overexpression of IGF-1 in a transgenic mouse model (15), and local infusion of IGF-1 to the rat tibialis anterior muscle (3). In the first study, Criswell and colleagues discovered that the mice overexpressing IGF-1 demonstrated a higher absolute muscle mass in both the gastrocnemius and the tibialis anterior (TA) compared to their wild-type counterparts. In the second study, the authors implicated IGF-1 in acting directly to stimulate protein synthesis and satellite cell proliferation, resulting in skeletal muscle hypertrophy. Further, these conclusions were supported by the observed increase in absolute weight of the TA injected with IGF-1. Additionally, the total protein and DNA content of these muscles increased. Both of these models produced dramatic muscle hypertrophy without any change in loading pattern, indicating the sufficiency of IGF-1 for skeletal muscle hypertrophy. Not only is IGF-1 necessary for skeletal muscle growth, its expression is increased during periods of increased load. Functionally overloaded rat plantaris muscles experienced a significant increase in IGF-1 mRNA as well as the compensatory increases in muscle size and fiber cross-sectional area (CSA) when compared to the contralateral control plantaris (78). Interestingly, these rats were hypophysectomized to decrease the levels of pituitary-derived growth hormone, illustrating the inherent importance of IGF-1s autocrine and paracrine properties to muscles during overload-induced hypertrophy. Regenerating skeletal muscle also shows an increase in the IGF-1 signal. Rat TA muscles recovering from bupivicaine injection demonstrated temporal and maximal expressions of IGF-1 mRNA at days 5 and 10, respectively (43). Further, Yang and

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12 colleagues (79) showed that IGF-1 is strongly correlated with the phenotypic adaptations associated with stretch and overload. Therefore, IGF-1 signaling is important for both skeletal muscle growth and the phenotypic changes associated with this growth. Adams and Haddad (2) suggested that the increases in muscle IGF-1 preceded the phenotypic changes in the functionally overloaded rat plantaris. These IGF-1 mRNA increases peaked at 3 days of overload with a maximal increase of 4 times normal levels. Finally, McCall and colleagues (44) showed that IGF-1 is transcriptionally regulated in vivo during hypertrophy as evidenced by the induction of the endogenous IGF-1 pre-mRNA during functional overload of the rat plantaris. IGF-1 induction in cell culture. In vitro studies have also been used to elucidate the pathways that participate in and activate muscle growth and the phenotypic changes associated with the growth. The research designs most commonly used are myotubes subjected to stretch or electrical stimulation. Media depleted of mitogens is commonly used to activate differentiation, as it induces the expression of localized IGF-1 (83). This induction overrides the negative control of serum mitogens on differentiation. Electrical stimulation (ES) produces changes in metabolic enzyme and contractile protein gene expression, and stretch potentiates the ES-induced changes, including harvesting a more robust IGF-1 response (46). Bayol et al.(6), suggest that proteins associated with the IGF-1 axis, in part, mediate these changes. Addition of IGF-1 to culture media stimulates both MAPK/ERK and PI3K/Akt signaling pathways during differentiation, but not to the same degree, suggesting the necessity of both pathways during proliferation and the induction of differentiation (22, 63). IGF-1 enhanced in vitro muscle growth through increased satellite cell proliferation via the PI3K/Akt pathway (12). Finally, repetitive

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13 stretch/relaxation of differentiated muscle cells stimulated the acute release of IGF-1 during the first 4 hours of stretch (51). In vivo and in vitro models suggest that IGF-1 uniquely stimulates both muscle cell proliferation and differentiation. MGF acts as a mechanically sensitive splice variant. Although IGF-1 is essential to muscle growth and maintenance, recent evidence suggests a splice variant of the growth factor that may be particularly responsive to loading and/or injury. Goldspink and colleagues (81) have identified and cloned this splice variant of IGF-1, called mechano growth factor (MGF). MGF, produced by active muscle, controls local tissue repair, maintenance, and remodeling through induction of local protein synthesis, and prevention of apoptosis. The alternative splicing of the IGF-1 mRNA results in a 52 base pair insert in the E domain that likely causes it to bind to a different protein. The reading frame shift associated with the MGF peptide also causes it to have a different C terminal sequence, inducing differential protein/receptor affinities. Therefore, the MGF peptide may mediate slightly different processes. In vitro, MGF inhibits terminal differentiation of C2C12 cells and increases cell proliferation by processes that are independent of the IGF-1 receptor (82). MGF expression increases with high resistance exercise in humans (30), following acute local damage in rat muscle (32), and with in vivo electrical stimulation and stretch (13). The peak in MGF expression is approximately on the third day of differentiation. Therefore, local production and post-transcriptional processing of IGF-1 is critical for adaptations in muscle mass in response to altered loading. Nevertheless, the signal(s) linking mechanical load and skeletal myofiber IGF-1 gene expression remain a mystery.

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14 Skeletal Muscle Hypertrophy and Vascular Endothelial Growth Factor (VEGF) Skeletal muscle hypertrophy is temporally coupled to the induction of angiogenesis and is closely linked to blood vessel recruitment and capillary angiogenesis, such that capillary density is maintained during the early stages of hypertrophy (54). Regular contractile activity induces an increase in capillarization. Along with basic fibroblast growth factor (bFGF), VEGF regulates angiogenesis, and is highly induced in growing and hypertrophying skeletal muscle (66). VEGF is a potent mitogen of endothelial cells. VEGF binds to two primary receptors on endothelial cells (VEGFR1 and VEGFR2). In addition to inducing endothelial cell proliferation, migration and differentiation, activation of VEGFR2 leads to NO production via eNOS. NO production can be vital to VEGF signaling, as inhibiting NO production diminishes the angiogenic response (7, 41). Recently, expression of VEGF in skeletal muscle cells has been shown to be under the control of IGF-1-dependent Akt signaling (66) and its expression is increased via an AMPK-p38 MAPK signaling cascade. VEGF expression and skeletal muscle overload. Increased angiogenesis is necessary to support a growing muscle as its metabolic demands increase. In humans (56), dogs (31), and rats (80), increased metabolic demands associated with an increased exercise paradigm caused enhanced remodeling of capillaries local to the muscle, and increased muscle contraction in rats caused an upregulation of VEGF (4). Although many models have induced angiogenesis by way of increases in blood flow and accompanying capillary shear stress, few have investigated the isolated role of muscle contraction and overload on growth factor expression and release, and on endothelial cell proliferation.

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15 Skeletal muscle overload is a potent stimulus for capillary growth in vivo. Rat extensor digitorum longus muscles exhibited an increase in capillary-to-fiber ratio after removal of the synergist tibialis anterior muscle (19), thereby implicating the importance of capillary angiogenesis during synergist ablation and the resulting overload. Degens and colleagues (17) found that VEGF mRNA expression increased after 2 weeks of compensatory overload following functional elimination of synergists to the plantaris, and this expression precedes capillary proliferation. This timepoint coincides with the half time for increases in capillary supply, suggesting VEGF plays a preparatory role in capillary angiogenesis and may be elevated during the week of recovery post surgery. Additonally, NO donors increase VEGF mRNA in vivo. Benoit and colleagues (7) found that injecting rats with nitroprusside, an NO donor, resulted in elevated levels of the growth factor in the gastrocnemius muscle. Further, another NO donor, S-nitroso-N-acetylpenicillamine (SNAP) induced angiogenesis in vivo and in vitro (41). Endothelial cells treated with SNAP demonstrated increased cell migration and differentiation into capillaries. Therefore, there is strong evidence that NO acts as a potential coordinator of capillary angiogenesis via transcriptional regulation of VEGF. VEGF and skeletal muscle cells. In addition to playing an important role in capillary angiogenesis, VEGF has also been implicated in stimulating skeletal muscle fiber regeneration in vivo. Arsic and colleagues (5) delivered VEGF using an adeno-associated virus vector to animals subjected to unilateral ischemia and muscle injury. As a result, VEGF delivery significantly reduced the damaged area and increased the number of regenerating fibers when compared to control muscle. The same investigators also found that VEGF increased the number, length, and nuclear content of

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16 differentiated C2C12 cells. VEGF decreased the rate of proliferation and promoted differentiation, a result that paralleled VEGFs ability to protect the differentiating cells from apoptotic death. Taken together, these data suggest the importance of investigating VEGF as a target for a possible therapeutic role in muscular disorders. VEGF receptors are members of a family of tyrosine kinase receptors that are expressed by a variety of tissues. Initially described on endothelial cells, recent data has shown the presence of VEGF receptors on hematopoietic and vascular smooth muscle cells. More important to the present study, others present evidence of VEGF receptor expression on cultured myoblasts and myotubes, and on skeletal myofibers isolated in culture. This helps to explain the close association of myocyte remodeling with angiogenesis. Hepatocyte Growth Factor (HGF) and Muscle Growth Satellite cells are skeletal muscle progenitor cells. Skeletal muscle satellite cells are myogenic stem cells residing between the sarcolemma and the basement membrane in postnatal skeletal muscle. Normally quiescent, they are activated in response to mechanical changes in the muscle. They actively participate in muscle regeneration following injury and are required for load-induced hypertrophy. Inactivation of satellite cells by gamma irradiation prevents muscle hypertrophy following compensatory overload. Once activated, the satellite cells migrate to the area of injury or growth to supply myonuclear precursors to the expanding fibers, preserving the myonuclear domain inherent to adult skeletal muscle. In response to stretch or compensatory overload, satellite cells are activated, enter the cell cycle and subsequently proliferate. Schiaffino and colleagues (61) have shown that this proliferative activity begins at ~3 days after

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17 compensatory overload. Activated satellite cells fuse into preexisting fibers or form new fibers (45). Although other growth factors have the capacity to stimulate satellite cell proliferation and/or differentiation (IGFs and fibroblast growth factors (FGFs)), it appears that hepatocyte growth factor (HGF) initiates satellite cell activation. It is active as a heterodimer that results from proteolytic cleavage of the inactive, single chain pro-form. Present in the liver, kidney, lung, and spleen, HGF message and protein have also been localized in adult, uninjured skeletal muscle sequestered to the extracellular matrix (62, 67). The signaling receptor for HGF is the c-met receptor, and both its message and protein have been found in satellite cells in vitro and in vivo. Muscle stretch or injury releases HGF from the extracellular matrix to bind the c-met receptor, thereby inducing the satellite cells to enter the cell cycle and proliferate. Cultured satellite cells also express HGF, which acts in an autocrine fashion to induce proliferation (62). Additionally, chronic low frequency stimulation of rat muscle in vivo leads to the activation and proliferation of satellite cells. Further, this increase in satellite cell progeny paralleled the increase in myonuclear content (55). However, the link between changes in external mechanical and electrical stimuli and chemical signaling is poorly understood. HGF release and compensatory overload. Overloaded plantaris muscle shows an increase in HGF message. After unilateral removal of the synergists to the plantaris, HGF mRNA expression increased at 3, 7 and 21 days when compared to the contralateral control (78). Plantaris hypertrophy was closely associated with the upregulation of HGF and FGF mRNA, even when the plantaris muscles were denervated during the overload

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18 period. This suggests that HGF expression may be involved in overload-induced hypertrophy. HGF and stretch. HGF release from the extracellular space and subsequent satellite cell activation appears to depend on external load. Cultured quiescent satellite cells subjected to stretch entered the cell cycle earlier than those in control conditions, and conditioned media from these stretched cells activated unstretched satellite cells (68). Additionally, blocking HGF in stretched cultures prevented satellite cell activation. In vitro experiments suggest that acute satellite cell activation is mediated by release of local HGF already present in skeletal muscle. NO serves as the link between mechanical stretch in vitro and HGF release from satellite cells. Tatsumi et al. (67) found that NOS activity is increased in cyclically stretched cells and the resulting NO mediates HGF release. HGF is synthesized and secreted by satellite cells in vitro, suggesting HGF possesses both autocrine and paracrine properties (62). Further, it is suggested that isolated satellite cells also express and secrete an HGF activator to ensure the active form of HGF will be available for autocrine action. Other progenitor cells, specifically fibroblasts, do not express HGF message or protein, lending support to the idea that satellite cells are the cells responsible for increasing HGF levels upon activation. Based on these data, it seems likely that sustained hypertrophic activity may require upregulation of HGF expression in activated satellite cells and/or muscle fibers Myogenin (MGN) and Control of Adult Muscle Phenotype The myogenic regulatory factors (MRFs), including MGN, MyoD, myf-5, and MRF-4, belong to the basic helix-loop-helix superfamily of transcription factors. These proteins form heterodimers with ubiquitous E-proteins and bind to DNA at consensus E

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19 box domains, present in many muscle-specific gene promoters and drives the differentiation process (52). During muscle development, the MRFs are expressed in a highly regulated temporal pattern, with MGN appearing late in differentiation corresponding to the beginning of myoblast fusion. MGN continues to be expressed in adult skeletal muscle fibers and is believed to control the slow-twitch fiber phenotype. MGN is an important regulatory factor during overload-induced hypertrophy. Its expression in activated satellite cells corresponds to terminal differentiation of these cells and fusion with existing muscle fibers, a process that is required for normal hypertrophy of adult muscle fibers. Hypertrophy and Nitric Oxide The exquisite coordination of the complex response to skeletal muscle overload suggests a common regulator. Smith et al. (64) reported that inhibition of nitric oxide synthase (NOS) activity prevents the normal hypertrophy and fiber type adaptations to chronic skeletal muscle overload. Further, the release of HGF during skeletal muscle loading is nitric oxide synthase-dependent (68), and endurance exercise increases VEGF expression in skeletal muscle via a nitric oxide-dependent mechanism (24). Together, these data suggest that nitric oxide may be acting as a common master signal. Therefore, we hypothesized that inhibition of NOS activity would prevent the increased local expression of growth/regulatory factor mRNAs (IGF-1, MGF, HGF, VEGF, and myogenin) and Type I (slow) myosin heavy chain (MHC) expected during the early stages of functional overload in the rat plantaris. NOS activity and fiber type composition. Adult skeletal muscle is a mosaic of four different fiber types, one slow (I), and three fast (IIa, IIb, IId/x). These differ with respect to specific biochemical and morphological characteristics. Skeletal muscle

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20 possesses a high degree of plasticity, allowing it to respond and adapt to altered physiological demands by switching between fiber types. The adaptation to functional demands is a well-documented process, known as fast-to-slow or slow-to-fast transformation. A fast-to-slow transformation is induced by muscle growth (hypertrophy), and results in an increased oxidative capacity and a greater resistance to fatigue. Conversely, a slow-to-fast fiber transformation characterizes muscle wasting (atrophy) and heightens a muscles fatigability and glycolytic capacity. Expression of the different fiber isoforms is determined by the type of nerve (8), the level of physical activity (60), and the amount of passive stretch (59). There has been extensive research in uncovering the pathways or molecules that are responsible for directing the fiber type transition, including NO. The metabolic phenotype of skeletal muscle fibers, including fiber type, is controlled primarily by calcium-dependent signaling. The tonic activity of slow motor neurons produces a chronic low-level of cytoplasmic calcium in slow-twitch fibers. This activates calcium-dependent signals. Most notably, the calcium-dependent phosphatase, calcineurin, and the calcium-calmodulin-dependent protein kinases (CaMK). The constitutive NOS enzymes are also calcium-calmodulin dependent and may play a regulatory or facilitative role in the control of muscle fiber type. Smith and colleagues (64) demonstrated the involvement of NOS in skeletal muscle adaptations to overload. As in previous research, overload induced hypertrophy and a fast-to-slow fiber type shift. Treatment with the NOS inhibitor, L-NAME, however, attenuated the overload-induced hypertrophy and prevented the increase in type I fibers with hypertrophy. This suggests that NO is necessary for changes in skeletal muscle phenotype in vivo. Additionally, NO

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21 covalently modifies thiol groups on the ryanodine receptors associated with calcium channels of the sarcoplasmic reticulum. This modification results in a release of calcium into the cytosol (21). Therefore, NO could facilitate activation of calcium-dependent pathways. Planitzer et al. (53) discovered that fast-oxidative fibers carry the highest concentration of NOS-1, possibly facilitating the fiber type transitions through a slower phenotype. Future research on NOS activation and the associated changes in phenotype is important in elucidating the complex signaling pathways associated with hypertrophy and muscle fiber type. Summary Disease states and conditions such as spaceflight and extended bed rest unload skeletal muscle and cause muscle atrophy. The characteristic phenotypic changes associated with muscle wasting are a serious clinical concern and modalities to prevent these changes should be investigated. Defining the mechanisms by which intracellular and extracellular signaling molecules control skeletal muscle growth shows promise for developing novel therapeutics to combat muscle wasting. The rat model of unilateral synergist removal provides a robust hypertrophic response, making this model a valuable tool in elucidating the regulatory pathways. Nitric oxide is a ubiquitous signaling molecule, located in essentially every tissue in the body. Inhibition of nitric oxide synthase (NOS) activity interferes with the muscle growth response. However, NOs specific role in muscle hypertrophy remains a mystery. Previous research implicates NO signaling in the fiber type transition associated with muscle growth. Further, NO plays a role in satellite cell activation and proliferation. Therefore, NO may act as a coordinating molecule in the many different growth steps of skeletal muscle. This project will

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22 determine whether blocking NOS activity can attenuate the expression of various growth/regulatory factors during functional overload of the plantaris muscle.

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CHAPTER 3 METHODS Ablation and Sham Sur g eries PBS L-NAME + PBS n=8 TRIM n=8 5-Day Overload Sacrifice & Tissue Harvestin g Sur g eries LN AME & TRIM 24 Female Sprague-Dawley Rats, 4month-old Day Day 0 Day 5 Figure 3-1. Experimental design flowchart. Animals were given the drugs starting 2 days before the surgeries. Tissue extraction was on day 5. Animals The subjects were adult (~4 month-old) female Sprague-Dawley rats (~250 g). All were housed in the J. Hillis Miller Animal Science Center and fed the same diet (rat chow and water ad libitum) throughout the experiment. They were maintained on a 12 h light:dark photoperiod (light 0700 to 1900h). All procedures followed NIH guidelines 23

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24 and were approved by the University of Floridas Institutional Animal Care and Use Committee. Inhibition of NOS Activity After an acclimation period of one week after arrival, the rats were randomly divided among the three experimental groups. The pharmacological inhibition of nitric oxide synthesis was achieved by administering the competitive non-isoform-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical) or the nNOS-selective inhibitor, 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM, Cayman Chemical) during the treatment period. L-NAME (1mg/ml) was added to the drinking water to maintain a dose of ~90 mg/kg/d. TRIM was dissolved in phosphate buffered saline (PBS) and injected intraperitoneally (IP) at a concentration of 10 mg/kg/d. To control for the possible confounding influences of a daily IP injection, the Control and L-NAME animals were injected daily with a volume of PBS equal to the volume injected into the TRIM animals. Synergist Ablation Surgery Chronic overload of the plantaris was induced by surgical, unilateral removal of the synergist muscles to the plantaris. The rats were anaesthetized with inhaled isoflurane (2-5%) with oxygen as the carrier gas. Using aseptic technique, a midline incision was made in the skin of the right hind limb, from the popliteal fossa to the Achilles tendon region. A second longitudinal incision was made through the hamstrings exposing the distal gastrocnemius and Achilles tendon region. The gastrocnemius tendon was carefully separated from the plantaris tendon, and the gastrocnemius muscle sectioned. The distal two-thirds of the gastrocnemius was removed, taking care not to disturb the plantaris

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25 nerve and blood supply. Next, the soleus muscle was carefully isolated and removed. The hamstring incision was closed with 4-0 vicryl absorbable suture. The overlying skin was closed with sterilized metal wound clips and treated with a topical antibiotic cream to avoid infection. A sham operation was performed on the left leg of each rat. In this procedure, the same incisions were made as above and the gastrocnemius tendons were isolated without transecting the muscles. The animals were allowed to fully recover from the anesthetic before returning to their cages. The rats were examined daily for signs of infections or wound openings, which were promptly treated, if found. Experimental Protocol Two days before the surgery, L-NAME was added to the drinking water of the appropriate group, and daily injections of TRIM (TRIM group) or PBS (Control and L-NAME groups) were begun. Forty-eight hours after the start of drug treatments, the animals underwent the ablation surgeries. All animals received the unilateral synergist ablation surgery along with the sham surgery on the contralateral limb. After a brief recovery period, the rats were group-matched, housed in pairs, and allowed to ambulate freely. Water was replaced each day, and body mass and water consumption were recorded daily throughout the experimental period. The dose of L-NAME was calculated for each rat. On day 5 post surgery, the animals were anesthetized with inhaled isoflurane and sacrificed by exsanguination. The plantaris muscles were immediately and bilaterally removed, trimmed of excess connective tissue and fat, weighed on an analytical scale, and flash-frozen in liquid nitrogen. Frozen muscles were powdered using a liquid nitrogen-cooled mortar and pestle. The powdered muscle was divided into separate tubes and stored at -80 C for subsequent biochemical analyses.

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26 Nitric Oxide Production To determine the efficacy of the pharmaceutical NOS blockers, L-NAME and TRIM, serum nitrate/nitrite levels were measured. Whole blood (~ 4 mL) collected from the animals at the time of sacrifice was incubated for 30 minutes at room temperature to allow for clotting. The samples were centrifuged 20 minutes at 5000g to separate the serum from the cellular fraction. The serum was removed and stored at -80C until further analysis. Using a pre-designed kit (Cayman Chemical), the serum samples were analyzed for nitrate/nitrite levels as per the manufacturers specifications. Reverse Transcription and Real-Time Quantitative PCR Total RNA was isolated using Trizol Reagent (Life Technologies, Carlsbad, CA) according to the manufacturers instructions. The amount of total RNA was evaluated by spectrophotometry and the integrity checked by gel electrophoresis. Total RNA (5 g) was reverse transcribed using the Superscript III First-Strand Synthesis System (Life technologies, Carlsbad, CA) using oligo(dT) 20 primers and the protocol outlined by the manufacturer. One L of cDNA (5 L for MGF) was added to a 25 L PCR reaction for real-time PCR using Taqman chemistry and the ABI Prism 7000 Sequence Detection System (Applied Biosystems (ABI), Foster City, CA). The comparative Ct method (ABI User Bulletin #2) was employed for the relative quantitation of gene expression. Hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as the normalizer. The enzyme is important in purine biosynthesis, and it was chosen based on initial experiments showing that our manipulations did not affect the expression of the transcript. Five-fold dilution curves were assayed on selected samples to confirm the validity of this quantitation method for each gene. IGF-1(GenBank NM_178866), HGF

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27 (GenBank NM_017017), skeletal -actin, Type I (slow) MHC, and myogenin (GenBank NM_017115) mRNA transcripts were assayed using pre-designed rat primer and probe sequences commercially available from Applied Biosystems (Assays-on-Demand). MGF (5-CACTGACATGCCCAAGACTCA (forward) and 5-CTTTGCAGCTTCCTTTTCTTGTG (reverse)) and HPRT (5-GTTGGATACAGGCCAGACTTTGT (forward) and 5-AGTCAAGGGCATATCCAACAACAA (reverse)) mRNA were assayed using custom made primers (Applied Biosystems, Assays-by-Design). The MGF reverse primer was custom designed to span the 52-base pair insert unique to the rat MGF cDNA. Semi-Quantitiave RT-PCR Because the pre-designed rat primers and probes supplied by ABI did not differentiate among the four isoforms of VEGF, we chose to do semi-quantitative PCR to determine if there was differential expression between the isoforms associated with this model. Total RNA was isolated as described above and reverse transcribed using Ready-to-Go You-Prime First-Strand Beads (Amersham Biosciences, Buckinghamshire, UK) and a combination of random hexamers and oligo(dT) 20 primers. cDNA for the four isoforms of VEGF expressed in rat skeletal muscle were amplified simultaneously using published primer sequences and conditions (16), with expected sizes of 632-, 560-, 500-, and 428-bp PCR products for amplification of VEGF-188, VEGF-164, VEGF-144 and VEGF-120, respectively. 18S ribosomal cDNA was amplified simultaneously as an internal control for amplification efficiency (Ambion, QuantumRNA internal standards). PCR products were separated by electrophoresis on 1% agarose gels, stained with ethidium bromide, and band intensity was quantified by densitometry (Scion Image software).

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28 Immunoblotting It has been shown that TRIM has significant inhibitory effects on both nNOS and the inducible form of NOS, iNOS. Both nNOS and eNOS are constitutively active in skeletal muscle, while iNOS activity is normally absent. However, iNOS protein expression and activity can be induced in skeletal muscle by the inflammatory process. To assess the possibility of the potentiation effects of iNOS, we evaluated iNOS protein levels via Western blotting in the normal loaded (NL) and the overloaded (OL) plantaris muscles from the Control group. For all Western blots, the powdered muscle was homogenized in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM -glycerol phosphate, 1 mM sodium orthovanadate, 1 g/ml leupeptin, 1 mM PMSF, and 10 g/ml aprotinin using a stainless steel blade. Homogenates were centrifuged for 10 min at 1000g to remove connective tissue and cellular debris and protein content quantified in the supernatant using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA, USA). For iNOS protein, 80 g of total protein were subjected to SDS-PAGE on 7% polyacrylamide gels. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes under cold conditions, which were stained with Ponceau S (0.1% w/v in 5% acetic acid) to verify equal loading. The membranes were subsequently blocked for 1h at room temperature in Tris-buffered saline-Tween (TBST; TrisHCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk and then incubated (4C overnight) with primary antibody for iNOS (Transduction Laboratories) diluted in blocking solution. The membranes were washed three times in TBST, 10 minutes each, followed by treatment with horseradish peroxidase-labeled anti-mouse antibody (Vector

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29 Labs) for 2h at room temperature. Reactions were developed using the Enhanced Chemiluminescence detection reagents (ECL; Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions, and protein levels were determined by densitometry (Scion Image software). For p70 S6K membranes were then blocked for 1 h in Tris-buffered salineTween (TBST; TrisHCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk and incubated with primary antibody for phosphor(Thr389)-p70 S6K (Cell Signaling Technology, Beverly, MA) overnight at 4C. Membranes were washed three times in TBST, 10 min each, followed by incubation with a peroxidase-labeled anti-rabbit antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at RT. Reactions were developed using the Enhanced Chemiluminescence detection reagents (ECL; Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions, and protein levels were determined by densitometry (Scion Image software). Limitations Although our lab has had previous success in using the synergist ablation model to induce overload hypertrophy, the procedure is not without criticism. The invasive nature of the surgery could affect skeletal muscle gene expression independent of mechanical overload. Nevertheless, comparison of overloaded muscles to contralateral normally-loaded muscles (i.e. within-subject design) controls for potential effects of systemic factors, such as circulating cytokines, hormones, or inflammatory cells. Further, sham surgeries control for local surgical effects. It is impossible to determine the source of intramuscular NO in the present design. Future studies will be necessary to examine potential intrinsic and extrinsic sources of NO signaling during muscle overload.

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30 Our model investigated the effects of 5 days of overload on gene expression of several growth factors, myogenin, and contractile proteins. This time point was chosen based on published accounts of overload-induced IGF-1 and MGN expression, protein synthesis, and RNA and DNA accumulation. All of these factors are significantly elevated in the overloaded plantaris at 5 days. However, the dynamics associated with HGF and VEGF induction is unknown, as is the time course of NO signaling. Therefore, more studies utilizing the functional overload model at different time points during the hypertrophic response may be necessary to elucidate possible differential gene expression. Vertebrate Animals Female Sprague-Dawley rats were used in this research. This study required the removal of hindlimb muscles synergistic to the plantaris via a non-terminal surgery, with subsequent ambulation. This invasive procedure prevented the use of human subjects. Sprague-Dawley rats were selected based on the large amount of preliminary data collected with this model in our lab and many others. Statistical Analysis This experiment was designed to test the hypothesis that NOS activity is necessary for the induction of growth factor and contractile protein expression during overload-induced hypertrophy. A 3 x 2 (treatment x loading condition) ANOVA with repeated measures on the loading condition was employed to determine main effects and interactions for each variable. Where significant differences were found, Tukeys HSD test was implemented post hoc to determine individual group differences. Significance was established a priori at p<0.05.

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CHAPTER 4 RESULTS Systemic and Biological Responses to Treatment Body mass did not change from preto post-overload treatment for any group. Further, body mass did not differ between groups at any time point (Table 4-1). Consistent with a report from Adams and Haddad (2), no OL-induced changes in total protein were observed at 5 days of overload. Nevertheless, this model does cause muscle protein accumulation following 14 days of OL (64). This study focuses on the early signaling events leading to this protein accumulation. Water consumption in the L-NAME-treated group was reduced ~40% during the first 24h of L-NAME treatment, but then returned to normal (not different from the Control group; data not shown) for the remainder of the treatment period. The average L-NAME dose was 89.2 mg/kg/d. L-NAME and TRIM treatments significantly lowered serum nitrate/nitrite levels (P<0.05), indicating successful systemic NOS inhibition. Mean (SEM) serum nitrate/nitrite levels were: Control = 7.08.31 M, L-NAME = 1.65.09 M, and TRIM = 4.49.19 M. Myogenin mRNA Expression Five days of plantaris overload caused an approximately 4-fold increase in myogenin mRNA expression. No significant differences were observed between treatment group (Table 4-2). 31

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32 Contractile Protein mRNA Expression Five days of OL resulted in a 90% increase in skeletal -actin mRNA and a 140% increase in type I (slow) MHC (MHC-I) mRNA (Figure 4-1; Control NL vs. Control OL). However, these data indicate a complete inhibition of this OL-induced response in the NOS-inhibited groups. Skeletal -actin and MHC-I mRNA levels did not differ between NO and OL muscle in either treatment group (L-NAME and TRIM). Further, skeletal -actin and MHC-I transcript levels in L-NAME and TRIM muscles did not differ from that in control NL muscles. Growth Factor mRNA Expression Expression of mRNA for HGF, IGF-1, MGF, and the 120 amino acid splice variant of VEGF (VEGF-120) were all increased in the OL muscles (Table 4-2). IGF-1 mRNA was increased ~4 fold and MGF mRNA ~9 fold in Control/OL compared to Control/NL muscles (Figure 4-2). TRIM treatment did not affect IGF-1 or MGF mRNA expression in the NL muscle, but approximately doubled expression of both transcripts in the OL muscle compared to Control/OL (Table 4-2). HGF and VEGF-120 mRNAs were increased 15-20 fold in the OL muscles compared to NL, with no effect of either L-NAME or TRIM treatment (Table 4-2 and Figure 4-3). Transcripts for VEGF-188, VEGF-164, and VEGF-144 tended to be reduced in the OL muscles, but this did not reach statistical significance (p>0.05). Real-time PCR assessment of total VEGF mRNA showed a ~50% reduction in OL muscle, compared to NL. The discrepancy between real-time assessment of total VEGF mRNA (Table 4-2) and semi-quantitative assessment of VEGF isoform expression (Figure 4-3) is most likely due to the relatively small contribution of VEGF-120 to the total VEGF mRNA pool and the variability in the semi

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33 quantitative RT-PCR method, which failed to demonstrate a significant OL-related reduction in the more abundant VEGF isoforms. Phosphorylation of p70 S6K Expression of total p70 s6k protein was increased in OL muscles in all three treatment groups. Likewise, phosphorylated p70 s6k was increased in OL muscles compared to NL. The ratio of phosphorylated to total p70 s6k did not differ between Control NL and OL muscles. Conversely, the ratio of phospho to total p70 s6k was significantly elevated in OL muscles from L-NAME and TRIM animals, indicating a greater relative phosphorylation status (Figure 4-4) in these muscles. iNOS Protein Expression TRIM significantly inhibits nNOS and, to a lesser degree, iNOS. Therefore, we sought to determine if iNOS induction during overload could contribute to the observed nitric oxide-dependent effects. iNOS protein was not detected in either normally loaded or overloaded plantaris muscle (Figure 4-5), suggesting that the TRIM effects are due to inhibition of nNOS activity. Table 4-1. Body mass, plantaris mass, and total protein data for the overloaded rats. Body Mass (g) Plantaris mass (mg) Total Protein (mg/muscle) NL 323.0 21.1 38.8 3.8 Control 340.4 8.9 OL 430.8 13.9 36.1 4.0 NL 303.3 6.1 40.9 2.4 L-NAME 334.4 11.5 OL 387.0 22.1 43.5 3.5 NL 342.5 19.3 36.5 3.7 TRIM 340.1 9.8 OL 385.0 20.4 41.8 4.6 Definition of abbreviations: L-NAME = N G -nitro-L-arginine methyl ester; TRIM = 1-(2-trifluoromethyl-phenyl)-imidazole; OL = 5-day overloaded plantaris; NL = contralateral normally loaded plantaris. Values represent means SEM.

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34 Table 4-2. Real-time PCR quantification of mRNA transcripts for selected growth factors and a regulatory gene in the plantaris muscle. Transcripts are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and expressed relative to the Control, normally loaded (NL) value, using the comparative Ct method. Control L-NAME TRIM Transcript NL OL NL OL NL OL MGN 1.38 0.32 5.28 0.81 a 1.09 0.33 3.58 0.39 a,b 0.89 0.16 3.68 0.79 a,b IGF-1 1.12 0.18 3.98 0.28 a 1.06 0.18 5.06 0.59 a,b 1.22 0.14 8.49 0.86 a,b,c MGF 1.07 0.33 8.84 0.78 a 0.73 0.12 11.32 3.05 a,b 0.99 0.29 22.03 6.31 a,b,c HGF 1.18 0.30 19.76 4.1 a 2.45 0.80 20.78 2.7 a,b 1.85 0.39 17.78 0.62 a,b total VEGF 1.22 0.27 0.58 0.09 a 1.68 0.52 0.68 0.14 ,b 1.94 0.50 0.52 0.08 a,b Definition of abbreviations: L-NAME = N G -nitro-L-arginine methyl ester; TRIM = 1-(2-trifluoromethyl-phenyl)-imidazole; OL = 5-day overloaded plantaris; NL = contralateral normally loaded plantaris; MGN = myogenin; IGF-1 = insulin-like growth factor-1; MGF = mechanosensitive growth factor; HGF = hepatocyte growth factor; VEGF = vascular endothelial growth factor. Values represent means SEM. a = Significantly different from Control NL, p<0.05. b = Significantly different from corresponding NL (within group), p<0.05. c = Significantly different from Control OL, p<0.05.

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35 2.5 A a N L 2.0 OL Sk. -Actin/HPRT mRNA (Relative to Control NL) 1.5 1.0 0.5 0.0 Control L-NAME TRIM B Figure 4-1. Real Time PCR assessment and quantification of contractile protein mRNA transcripts. A) skeletal -actin mRNA level relative to HPRT mRNA in 5-day overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of Control, L-NAME and TRIM-treated rats. B) Type I (slow) myosin heavy chain (MHC) mRNA level relative to HPRT mRNA in 5-day overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of Control, L-NAME and TRIM-treated rats. Values represent means SEM.a = Significantly different from corresponding NL, p<0.05. Type I (slow) MHC/HPRT mRNA (Relative to Control NL) Control L-NAME TRIM N L OL a 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

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36 Figure 4-2. Real Time PCR quantification of insulin-like growth factor mRNA transcripts. A) Insulin-like growth factor-1 (IGF-1) mRNA level relative to HPRT mRNA. B) Mechano growth factor (MGF) mRNA level relative to HPRT mRNA. Values represent means SEM. a = Significantly different from Control/NL, p<0.05. b = Significantly different from Control/OL, p<0.05. Control L-NAME TRIM N L OL a,b a a 12 10 8 6 4 2 0 A IGF-1/HPRT mRNA (Relative to Control NL) B L-NAME TRIM Control a a a,b OL NL 30 25 20 15 10 5 0 MGF/HPRT mRNA (Relative to Control NL)

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37 A N L Figure 4-3. Semi-quantitative RT-PCR analysis of VEGF mRNA splice variant expression. A) Expression levels of VEGF-120 mRNA, relative to ribosomal 18S RNA in 5-day overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of Control, L-NAME and TRIM-treated rats. B) Representative ethidium bromide stained 1% agarose gel illustrating PCR products following amplification of VEGF and 18S. See Methods for details of assay conditions. Values represent means SEM. a = Significantly different from Control/NL, p<0.05. VEGF-120 mRNA/18S RNA (Relative to Control NL) a 25 OL a 20 a 15 10 5 0 Control L-NAME TRIM L-NAME Control TRIM B NL OL NL OL NL OL VEGF 188 VEGF 144 VEGF 164 VEGF 120 18S

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38 Figure 4-4. Western blot analysis of p70 s6K A) Representative immunoblot for phosphor(Thr389)-p70 s6K Totalp70 s6K and beta-actin (loading control) in 5-day overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of Control, L-NAME, and TRIM-treated rats. B) Quantification of phosphor(Thr389)-p70 s6K to total p70 s6K ratio. Values are means (SEM) expressed relative to Control/NL mean. a = Significantly different from corresponding NL p<0.05. b = Significantly different from Control-OL, p<0.05. MW Markers 80 kDa 50 kDa 80 kDa 50 kDa 39 kDa NL NL NL OL OL Control L-NAME OL TRIM A Phos-p70 s6 k Total-p70 s6 k Beta-actin B a,b 10.0 Phos p ho/Total p 70 s6 k Ratio ( Normalized to Control-NL ) 8.0 N L 6.0 OL a,b 4.0 a 2.0 0.0 Control L-NAME TRIM

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39 NL OL + control NL OL iNOS Figure 4-5. Immunoblot assessment of iNOS protein expression in 5-day overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of Control rats. iNOS expression was not detected in any of the samples.

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CHAPTER 5 DISCUSSION To our knowledge, this is the first study to investigate the effects of functional overload on the up-regulation of key signaling pathways leading to increased protein synthesis, angiogenesis, and satellite cell activation and proliferation. Our primary observations were focused on an early time point (5d of overload) when growth factor expression is at its peak (2). The data supports our hypothesis that NOS activity is important for up-regulation of contractile gene expression. Specifically, skeletal -actin type I (slow) MHC up-regulation in the overloaded plantaris was prevented with both non-isoform-specific inhibition of NOS activity (L-NAME) and nNOS-specific inhibition (TRIM). Conversely, neither L-NAME nor TRIM treatment repressed the overload-related increase in skeletal muscle mRNA expression for myogenin and the growth factors: HGF, VEGF-120, IGF-1, and MGF. In fact, the TRIM-OL group expressed approximately double the MGF and IGF-1 transcripts compared to Control-OL. Consistent with increased IGF-1 signaling, NOS inhibition induced greater relative phosphorylation of p70 s6K in OL muscle. Although it appears that nitric oxide signaling is not necessary for the up-regulation of the growth factors we measured, the data suggest that nitric oxide may play a role in the transcriptional regulation of slow MHC, skeletal -actin, the phosphorylation status of p70 s6k and, perhaps, feedback control of IGF-1/MGF mRNA during skeletal muscle overload. Employing in vivo models to investigate nitric oxide signaling, although physiologically meaningful, is not without consequence. Systemic non-isoform-specific 40

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41 NOS inhibition (L-NAME) has physiological consequences throughout the body, and can lead to significant effects on hemodynamics (14), muscle contractility (20), and gene expression in non-muscle tissue (35). The majority of these systemic L-NAME effects are thought to be secondary to eNOS inhibition and the resulting effects on blood flow and blood pressure. In fact, the nNOS-specific isoform, TRIM, has been administered to rats in vivo with no reported systemic side-effects (39). Since nNOS is the most abundant NOS isoform in skeletal muscle (70), and is reportedly sensitive to muscle loading (71), we hypothesized that this isoform accounts for overload-induced nitric oxide signaling in the plantaris muscle. To test this possibility, and partially control for the systemic effects of L-NAME, we treated one group of rats with daily IP injections of TRIM. Since TRIM inhibits iNOS as well as nNOS, we confirmed by immunoblots that iNOS protein was not expressed in NL or OL plantaris muscles (data not shown). Our data support a role for the nNOS isoform in early adaptations to skeletal muscle overload. Skeletal -Actin mRNA Expression Skeletal -actin protein is an important component of the contractile apparatus, and is known to be transcriptionally up-regulated during skeletal muscle hypertrophy(9). Since adult skeletal muscle sarcomeric actin is derived from the single -skeletal actin gene, rather than from multiple isoforms, the regulation of this gene serves as an index of overall contractile protein synthesis. Carson et al(11) have reported that transcriptional activity of the actin promoter is increased in skeletal muscle during in vivo stretch overload. This effect is mediated by serum response factor (SRF) binding to actin promoter (10). A recent paper (36) found that nitric oxide donors were sufficient to induce SRF binding to a myosin heavy chain promoter element and increase promoter

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42 activity in cultured smooth muscle cells. Our data suggest that nNOS activity is important for induction of skeletal -actin transcription during chronic overload. VEGF Expression The reduction in total VEGF mRNA level in the 5d-overloaded muscle was unexpected, given the known capillary angiogenesis occurring in overloaded rat muscle (54). However, the few studies reporting VEGF mRNA expression in hypertrophying skeletal muscle show mixed results. Degens et al. (17) found no significant changes in VEGF mRNA in hypertrophying quail muscle. Similarly, 4 wks of strength training in human subjects, even under hypoxic conditions, did not change skeletal muscle VEGF mRNA expression (23). On the other hand, overload of the rat plantaris for 2 wks (i.e. 2.8X longer than our 5d treatment) did increase VEGF mRNA by ~50% (18). To further characterize the VEGF mRNA response, we examined expression of the four splice variant isoforms found in rat skeletal muscle (16) using semi-quantitative RT-PCR and published primer sequences (16). To our knowledge, ours is the first study to measure expression of specific VEGF mRNA splice variants in overloaded, hypertrophying skeletal muscle. Unlike aerobic exercise, which primarily induces the VEGF-164/5 isoform (29, 37), we found that the VEGF-120 isoform was induced in the 5d-overloaded plantaris muscle. This effect, however, was not influenced by L-NAME or TRIM treatments. IGF-1 Expression and Phosphorylation of p70 s6 Kinase. These results are consistent with previous findings that endogenous IGF-1 mediates adult skeletal muscle hypertrophy, as our data shows a dramatic increase in the amount of transcript with OL. Additionally, we show that the MGF splice variant is particularly responsive to overload. These data support a role for nitric oxide that is either

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43 independent of the IGF-1 axis, or downstream of IGF-1 transcription. The exaggerated expression of IGF-1 and MGF mRNA in the overloaded plantaris of the nNOS-inhibited TRIM group suggest the possibility of an nNOS-dependent negative feedback mechanism controlling the load-induced IGF-1 response. IGF-1/Akt signaling in the rat kidney is known to activate eNOS by phosphorylation and increase nitric oxide production (75). The phosphorylation status of the NOS isoforms in overloaded skeletal muscle is unknown, but it seems possible that IGF-1-dependent nNOS activation could produce a feedback signal to control IGF-1/MGF expression during muscle growth. Activation of the key translational regulator, p70 s6K is correlated to increased protein synthesis in skeletal muscle, induced by phosphoinositide 3-kinase (PI3K) or mechanical stretch (33). Since NOS inhibition reduces protein accumulation in overloaded rat plantaris, we postulated that phosphorylation of p70 s6K would be inhibited in the L-NAME and TRIM groups. On the contrary, we found that OL induced expression of total p70 s6K protein in all groups, and that this corresponded to an increase in phosphorylated p70 s6K in the OL muscles. However, the ratio of phosphor/total p70 s6K indicating the relative degree of activation of the pathway was elevated only in the OL muscles of the L-NAME and TRIM groups. This suggests that protein translation may have been elevated in partial compensation to reduced transcriptional activity. Further experiments are needed to directly measure effects of nitric oxide on skeletal muscle protein synthesis rates during hypertrophy. Future Directions Although many of our results are unremarkable, there remain many unanswered questions regarding the mechanisms underlying skeletal muscle hypertrophy. Therefore, there are other pathways yet to be investigated that may play an important role in

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44 contributing to muscle growth. We will look at two possible contributors that deserve future attention and the possibility of interaction with nitric oxide signaling: calpain-mediated proteolysis and calcineurin-NFAT pathway. Calpain-Mediated Proteolysis. The calpains are a ubiquitous family of calcium-dependent cytosolic cysteine proteases. Calpain proteolysis activity contributes to overall protein degradation. Specifically, the calpains target proteins that are important in linking cytoskeletal proteins together to the cell membrane (25). Nitric oxide has been implicated as a possible regulator of calpain activity. The nitric oxide donor, sodium nitroprusside, reversibly inactivates calpain activity via S-nitrosylation (47). Further, NOS activity has been shown to inhibit calpain protease activity in skeletal muscle(38). Therefore, it is also possible that protein accumulation during overload in the NOS-inhibited groups could be hindered by an increase in protein degradation. Preliminary data comparing protein content of cleaved (II)-spectrin between the groups at 5 days of overload showed inconsistent results (data not shown). However, this could be due to a variety of factors, including length of time between tissue harvest and data collection in this instance. Nonetheless, calpain activity inhibition may represent an important mechanism by which nitric oxide production facilitates hypertrophy. Calcineurin-NFAT pathway and the slow MHC phenotype. Calcineurin is a calcium-dependent protein phosphatase located in skeletal muscle cytoplasm that preferentially responds to intracellular calcium concentration. Once activated, the cascade of events leads directly to muscle growth and fiber type differentiation (65, 73). A target for calcineurin is a member of the nuclear family of activated T-cells (NFAT), which remains in the sarcoplasm in a phosphorylated state. Upon dephosphorylation, the

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45 protein translocates to the nucleus and promotes transcription of genes which are involved in hypertrophy and fiber morphology shift (40, 50). Calcineurin has been implicated in both stimulation of type I gene expression and facilitation of type II to type I fiber type transition. Naya and colleagues (49) concluded that activated calcineurin induces type I gene expression via NFAT transcription. The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in the rephosphorylation and subsequent resequestering of NFAT to the cytoplasm. Interestingly, in cardiac muscle there exists a cross talk between JNK and calcineurin-NFAT signaling such that JNK activation acts to modulate calcineurin-NFAT signaling and inhibit cardiac growth(42). Further, nitric oxide increases NFAT nuclear accumulation indirectly via cGMP-dependent kinase (PKG) in smooth muscle (27). PKG, in turn, directly inhibits JNK activity, allowing NFAT to accumulate in the nucleus. Taken together, these data suggest additional pathways through which nitric oxide may be regulating the hypertrophy response in skeletal muscle and should be investigated further. Conclusions Up-regulation of type I (slow) MHC and skeletal -actin mRNA (presumably via transcription) during chronic skeletal muscle overload is dependent upon nNOS activity. Conversely, induction of growth factors and activation of protein translation (p70s6k phosphorylation) are not dependent upon NOS activity. Nevertheless, nitric oxide production may provide feedback control of IGF-1 and MGF signaling in hypertrophying muscle.

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LIST OF REFERENCES 1. Adams, G. R. Role of insulin-like growth factor-I in the regulation of skeletal muscle adaptation to increased loading. Exerc Sport Sci Rev 26: 31-60, 1998. 2. Adams, G. R., and F. Haddad. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 81: 2509-16, 1996. 3. Adams, G. R., and S. A. McCue. Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84: 1716-22, 1998. 4. Amaral, S. L., P. E. Papanek, and A. S. Greene. Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol Heart Circ Physiol 281: H1163-H1169, 2001. 5. Arsic, N., S. Zaccigna, and L. Zentilin. Vascular endothelial growth factor stimulates skeletal muscle regeneration in vivo. Molecular Therapy 10: 844-854, 2004. 6. Bayol, S., C. Brownson, and P. T. Loughna. Electrical stimulation modulates IGF binding protein transcript levels in C2C12 myotubes. Cell Biochem Funct [Epub ahead of print], 2004. 7. Benoit, H., M. Jordan, H. Wagner, and P. D. Wagner. Effect of NO, vasodilator prostaglandins, and adenosine on skeletal muscle angiogenic growth factor expression. J Appl Physiol 86: 1513-1518, 1999. 8. Buller, A. J., and J. C. Eccles. Interactions between motorneurons and muscles in respect of the characteristic speeds of their responses. J Physiol 150: 417-439, 1960. 9. Carson, J. A., D. Nettleton, and J. M. Reecy. Differential gene expression in the rat soleus muscle during early work overload-induced hypertrophy. FASEB J 16: 207-9, 2002. 10. Carson, J. A., R. J. Schwartz, and F. W. Booth. SRF and TEF-1 control of chicken skeletal alpha-actin gene during slow-muscle hypertrophy. Am J Physiol 270: C1624-33, 1996. 11. Carson, J. A., Z. Yan, F. W. Booth, M. E. Coleman, R. J. Schwartz, and C. S. Stump. Regulation of skeletal alpha-actin promoter in young chickens during hypertrophy caused by stretch overload. Am J Physiol 268: C918-24, 1995. 46

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BIOGRAPHICAL SKETCH Jeff Sellman was born in Kettering, Ohio, and grew up in Littleton, Colorado. He graduated summa cum laude from Heritage High School in 1991. After 2 years of undergraduate work at the University of Colorado at Boulder, Jeff took time off to pursue other interests. This led him to the Florida Army National Guard where he was a land combat missile system repair technician. During his time in the army, Sergeant Sellman was an expert M16 marksman, was designated an army physical fitness leader, and received his combat lifesaver certificate. He was awarded The Army Reserve Component Achievement Medal and The Army Achievement Medal. Jeff returned to school at the University of Florida, Gainesville, Florida, and graduated cum laude with a bachelors degree in exercise and sports sciences. He began a masters program in applied physiology and kinesiology also at the University of Florida. He has worked for four years as a research assistant in the Molecular Physiology Laboratory within the Center for Exercise Science. Jeff has been accepted into the University of Florida College of Medicine Class of 2009 and will pursue an M.D. degree. 54


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Title: Nitric Oxide Synthase Activity Affects Gene Expression in Overloaded Skeletal Muscle
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Copyright Date: 2008

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NITRIC OXIDE SYNTHASE ACTIVITY AFFECTS GENE EXPRESSION IN
OVERLOADED SKELETAL MUSCLE

















By

JEFF E. SELLMAN


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


2005





























Copyright 2005

by

JeffE. Sellman


































To my parents, Richard and Rebecca Sellman, for their unconditional love and limitless
support















ACKNOWLEDGMENTS

First and foremost, I would like to thank my parents (Rich and Rebecca Sellman)

for their unconditional and continued support. They have been both my life preserver

and my kayak when I needed help in keeping my head above water. Without their love, I

would run the risk of losing sight of the bigger picture.

This project was a part of Dr. David Criswell's vision of his fledgling laboratory.

As my committee chair and mentor, Dr. Criswell showed limitless patience, direction,

and faith in entrusting me with this project. I thank him for supplying me with the tools

(both tangible and not) to see the project to completion.

I acknowledge all of the graduate students and professors in the Center for Exercise

Science who witnessed my progression from an undergraduate student to a graduate

assistant. I especially thank Keith DeRuisseau for his knowledge of and input into my

results.

Finally, I thank Dr. R. Andrew Shanely and the Frank Booth laboratory for

technical assistance in running the assays.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ......... .................................................................................... iv

LIST OF TABLES ............. ..... ......................... .......... ............. vii

LIST OF FIGURES ............. ................... ............ .......... ............... .. viii

ABSTRACT ........ .............. ............. ...... ...................... ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

B background ............................................................... .. ........ ...............
Problem Statem ent .................. ........................................ ................ .2
H ypotheses ................................................ 3
Definition of Terms ....................... ...................................3
Limitations/Delimitations/Assumptions ........................................ ...............
Significance of the Stu dy .............................................................................. ............ 7

2 LITER A TU R E REV IEW ............................................................. ....................... 8

Skeletal M uscle A daptations to Load.................................... .................................... 8
Hypertrophy and Fiber Type Shifts with Loading....................... .... ..............9
Skeletal Muscle Hypertrophy and Insulin-Like Growth Factor (IGF-1) Signaling ...10
Skeletal Muscle Hypertrophy and Vascular Endothelial Growth Factor (VEGF) .....14
Hepatocyte Growth Factor (HGF) and Muscle Growth...........................................16
Myogenin (MGN) and Control of Adult Muscle Phenotype............... ................ 18
H ypertrophy and N itric Oxide ........................................................ ............. 19
S u m m a ry ............................................................................................................... 2 1

3 M E T H O D S ........................................................................................................... 2 3

A n im a ls ................................................................................................................. 2 3
Synergist Ablation Surgery.......... ......................... ........................... 24
E xperim mental P protocol ........................................................................ .................. 2 5
Nitric Oxide Production................................................................26
Reverse Transcription and Real-Time Quantitative PCR..........................................26
Sem i-Q uantitiave R T -P C R .............................................................. .....................27









Im m u n o b lo ttin g ................................................................................ ................ .. 2 8
L im station s ............. ................. .................................................................... 2 9
V ertebrate A nim als............. ................................................................ ........ ..........30
S tatistic al A n aly sis................................................................................................ 3 0

4 R E S U L T S ............. ..... ............ ................. .................................................. 3 1

Systemic and Biological Responses to Treatment.....................................................31
M yogenin m R N A Expression....................................................................... ........ 31
Contractile Protein mRNA Expression .............................................. ...............32
G row th Factor m RN A Expression............................................... ...................... ..... 32
P hosphorylation of p70S6K ............................................ ........................................33
iN O S P rotein E expression ......................................... .............................................33

5 DISCUSSION ............... ......... .................. 40

Skeletal a-Actin mRNA Expression ................ ........... ... .................... 41
VEGF Expression ............... ........ ....... ... ........................ .....42
IGF-1 Expression and Phosphorylation of p70s6 Kinase............................ ........42
F utu re D direction s ................................................................4 3
C o n c lu sio n s........................................................................................................... 4 5

LIST OF REFEREN CES ........................................ ........................... ............... 46

B IO G R A PH IC A L SK E TCH ..................................................................... ..................54
















LIST OF TABLES

Table page

4-1 Body mass, plantaris mass, and total protein data for the overloaded rats..............33

4-2 Real-time PCR quantification of mRNA transcripts for selected growth factors
and a regulatory gene in the plantaris muscle. ................................. ............... 34
















LIST OF FIGURES


Figure p

3-1 Experim ental design flow chart.................................................................... ....... 23

4-1 Real Time PCR assessment and quantification of contractile protein mRNA
transcripts.. ......................................... ............................... 35

4-2 Real Time PCR quantification of insulin-like growth factor mRNA transcripts.....36

4-3 Semi-quantitative RT-PCR analysis of VEGF mRNA splice variant expression....37

4-4 W western blot analysis of p7 6K..................... .... ..................................................... 38

4-5 Immunoblot assessment of iNOS protein expression.. ....................... 39















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

NITRIC OXIDE SYNTHASE ACTIVITY AFFECTS GENE EXPRESSION IN
OVERLOADED SKELETAL MUSCLE

By

JeffE. Sellman

December 2005

Chair: David Criswell
Major Department: Applied Physiology and Kinesiology

Nitric oxide is a mechanically sensitive signal in skeletal muscle. Inhibition of

nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat

plantaris. We investigated the mechanism for this effect by examining early events

leading to muscle growth after 5 days of functional loading. We also tested the

hypothesis that NOS activity is necessary for functional overload-induced upregulation of

growth factors, myogenin, and contractile gene mRNAs in the rat plantaris muscle.

Twenty-four female Sprague-Dawley rats (-250 g) were randomly divided into three

groups (n=8/group): Control, N-nitro-L-arginine methyl ester (L-NAME: 100 mg/kg/d),

or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM: 10 mg/kg/d). Unilateral removal of

synergists induced chronic overload (OL) of the right plantaris for 5 days. Sham surgery

was performed on the left hindlimb, which served as a normally loaded (NL) control. No

group differences were observed among NL muscles. Real-time PCR analyses showed

elevated (p<0.05) mRNA expression for insulin-like growth factor-1 (IGF-1), mechano-









growth factor (MGF: load-sensitive splice variant of IGF-1), hepatocyte growth factor

(HGF), and myogenin and reduced (p<0.05) total VEGF mRNA expression in the OL

muscle compared to NL. Neither L-NAME nor TRIM affected HGF, VEGF, or

myogenin responses. However, OL-induction of IGF-1 and MGF mRNA was greater

(P<0.05) in the TRIM group compared to the Controls. Conversely, overload-induction

of phosphorylated p70 S6 kinase (p70s6K) was prevented in the TRIM group. Type I

(slow) myosin heavy chain (MHC) and skeletal a-actin mRNAs were increased in the

Control/OL muscle (an effect that was completely prevented in both NOS-inhibitor

groups). Therefore, nNOS activity is necessary for overload-induction of Type I (slow)

MHC and skeletal a-actin mRNA and p70S6K phosphorylation. Further, the inhibition of

nNOS causes a compensatory increase in IGF-1 expression during overload.














CHAPTER 1
INTRODUCTION

Skeletal muscle is an extremely plastic tissue. Variations in external load result in

structural, biochemical, and morphological adaptations in muscle fibers. Specifically,

alterations in mitochondrial number, enzymatic profile, structural protein expression and

content, and capillary angiogenesis accompany both muscle atrophy and hypertrophy.

Chronic overload causes dramatic muscle growth. This hypertrophy response involves

the activation and later fusion of satellite cells to the muscle fibers (57, 58), and increased

synthesis of structural and contractile proteins (28, 76). Concurrently, capillary

angiogenesis is induced to support growing muscle (54). The increased fiber recruitment

associated with muscle overload also causes an up-regulation of slow fiber type-specific

genes. Molecular regulation of growth factors and transcription factors that govern

muscle growth is poorly understood. Therefore, discovering the molecules responsible

for signaling this coordinated response is vital for understanding load-induced adaptive

changes in skeletal muscle.

Background

Loss of skeletal muscle mass is a serious clinical problem in disease states (such as

cancer and AIDS) and in conditions such as prolonged bed rest and spaceflight.

Preserving muscle mass by attenuating muscle loss and/or stimulating muscle growth can

be vital to decreasing recovery time and increasing the patient's ability to ambulate freely

and enjoy independence. Muscle growth in response to increased load is a complex event

that requires the transcription of many different factors to contribute to the changing









phenotype; therefore, it is important to understand the underlying mechanisms

responsible for the transduction of mechanical forces into chemical signals. Nitric oxide

(NO) is an important signaling molecule, playing a role in various physiologic processes.

These effects are broad and far-reaching, as NO has been implicated in preserving

endothelial function and may be necessary for the slow fiber-type transition associated

with overload-induced hypertrophy. A family of nitric oxide synthases (NOS), all of

which can be expressed in adult skeletal muscle, governs its synthesis. Neuronal NOS

(nNOS) is the most abundant isoform in skeletal muscle, being associated with the

dystrophin complex (39, 71). The activity of this enzyme is induced by contractile

activity (70).

Problem Statement

Increased muscle load results in increased levels of growth/regulatory factors and

contractile proteins, and greater nNOS activity. Therefore, we postulate that the events

are related. We suggest that NO acts to coordinate the events associated with muscle

growth. We tested the effects of NOS inhibition on short-term responses to chronic

muscle overload.

Variables in Study

Independent variables. We manipulated load to the plantaris and NOS inhibition

by administering N-nitro-L-arginine methyl ester (L-NAME) and

1-(2-trifluoromethyl-phenyl)-imidazole (TRIM).

Dependent variables. We measured expression of growth factors (insulin-like

growth factor-1 (IGF-1), mechanogrowth factor (MGF), vascular endothelial growth

factor (VEGF), and hepatocyte growth factor (HGF)); a myogenic regulatory factor,









myogenin (MGN); and contractile proteins, skeletal a-actin and Type I (slow) myosin

heavy chain (MHC)

Control variables. Gender was excluded from the study: we used only female

Sprague-Dawley rats. Aging effects were also excluded: we restricted observations to

young adult rats (-4 months old).

Extraneous variable. We did not control for differences in the activities of the

animals. Since some animals may ambulate more freely than others, the load frequency

may differ among animals. However, preliminary experiments show a robust and

reproducible hypertrophy of the plantaris in response to unilateral ablation of its

synergists. Acute IP injections have the potential to induce a stress response that then

results in a cascade of proteins that could confound the study. To control for this, each

animal in the study was given an IP injection, either of TRIM or vehicle.

Hypotheses

1. Five days of functional overload will increase the local expression of
growth/regulatory factor mRNA's (IGF-1, MGF, HGF, VEGF, and MGN) and the
local expression of skeletal a-actin and Type I (slow) MHC.

2. NOS inhibition will prevent increased local expression of the growth/regulatory
factors, skeletal a-actin, and Type I (slow) MHC.

3. Specific nNOS inhibition will prevent increased local expression of
growth/regulatory factors, skeletal a-actin, and Type I (slow) MHC to the same
extent as non-isoform specific systemic NOS inhibition.

Definition of Terms

1-(2-trifluoromethyl-phenyl)-imidazole (TRIM): a selective inhibitor of the

neuronal isoform of NOS (nNOS).

Hepatocyte growth factor (HGF): an autocrine/paracrine factor that binds to the

c-met receptor. In skeletal muscle, HGF coordinates the activation and proliferation of









satellite cells during muscle regeneration and growth. This factor has been identified as

the component of crushed muscle extract that is capable of activating quiescent satellite

cells in vitro and in vivo (67).

Hypoxanthine guanine phosphoribosyl transferase (HPRT): an enzyme that

plays a role in purine biosynthesis. HPRT was chosen in this model as a normalizing

gene in Real-Time PCR because of its constant expression with our manipulations

(overload and NOS inhibition).

Insulin-like growth factor-1 (IGF-1): produced by the liver and skeletal muscle,

this growth factor is an important autocrine/paracrine factor capable of stimulating

proliferation and differentiation of activated satellite cells. IGF-1 also activates protein

synthesis in skeletal muscle fibers via Akt/mTOR activation of protein translation.

Mechanosensitive growth factor (MGF): a splice variant of IGF-1 that is

particularly sensitive to load and injury in skeletal muscle.

Myogenin (MGN): a myogenic regulatory factor that directs muscle

differentiation. MGN expression in adult muscle fibers plays a role in directing the slow-

oxidative phenotype.

NG-nitro-L-arginine methyl ester (L-NAME): a non-isoform selective

competitive inhibitor of nitric oxide synthase activity.

Nitric oxide (NO): rapidly diffusing signaling molecule synthesized by a family

of isozymes, the nitric oxide synthases. Its responses are largely mediated by S-

nitrosylation of cysteine residues or by interactions with iron and copper. It has been

implicated in many skeletal muscle processes, including gene expression.









Nitric oxide synthase (NOS): a complex enzyme responsible for the production

of NO. It acts on molecular oxygen, arginine, and NADPH to produce NO, citrulline,

and NADP+. The process requires five cofactors (FAD, FMN, Heme, calmodulin, and

tetrahydrobiopterin) and two divalent cations (calcium and heme iron). Three distinct

forms of NOS have been identified: nNOS, eNOS, and iNOS. All NOS isoforms are

expressed in mammalian skeletal muscle.

Normal load (NL): the contralateral (left) plantaris serves as an internal control

since the gastrocnemius, plantaris, and soleus remain intact. It is assumed that the work

done by the NL plantaris mimics the normal, physiologic condition.

Overload (OL): the right plantaris serves as the unit that receives a higher external

load due to removal of the muscles synergistic to it, the gastrocnemius and the soleus.

Polymerase chain reaction (PCR): a technique that creates copies of specific

DNA fragments by supplying gene-specific primers, a nucleotide pool, and DNA Taq

polymerase.

Reverse transcription (RT): a process that synthesizes DNA from an RNA

template (isolated total cellular RNA) using the enzyme reverse transcriptase as a

catalyst.

Satellite cells: normally quiescent, satellite cells are committed stem cells of adult

skeletal muscle. Residing between the basal lamina and the sarcolemmal surface of adult

muscle fibers, they are activated by conditions such as increased load and injury. They

help repair skeletal muscle and mediate muscle growth by differentiating into myocytes.

Vascular endothelial growth factor (VEGF): a regulator of blood vessel

synthesis induced by IGF-1 during muscle growth.









Limitations/Delimitations/Assumptions

Limitations. The invasive nature of this research negates the use of humans as

subjects. A rat model was chosen due to genetic homogeneity that allows the researcher

to draw conclusions about a population based on the sample studied. Also, rat skeletal

muscle shows similarities in phenotype and genetic makeup to human muscle.

Surgically induced overload provides a dramatic response in determining changes

in gene expression and alterations in protein synthesis. Therefore, this model provides

ease with which the researcher is able to uncover the signaling pathways responsible for

the changes associated with hypertrophy. However, unilateral removal of synergists may

not guarantee the animal utilizes each leg uniformly during post-surgical ambulation.

Therefore, it is possible that the contralateral limb serving as the NL control may

experience a small hypertrophic response should the animal favor that leg. To control for

this, we performed a sham surgery to the left leg by isolating the synergists without

transaction. Therefore, the animal would perceive injury to both legs that would allow

the ambulation to better mimic the normal, physiologic condition.

Similar to previous studies using L-NAME administered in the animal's drinking

water, our animals showed an initial decrease in the water consumed. However, after an

adjustment period, the L-NAME animals did not consume liquid at a different rate than

either the TRIM or the Control groups.

Finally, TRIM administered intraperitoneally (IP) may have induced an undesired

stress response in the animal. Therefore, the L-NAME and Control animals received IP

injection of phosphate-buffered saline (PBS) vehicle. TRIM inhibits both nNOS and, to a

lesser extent, iNOS. Therefore, to assess the possible potentiation effects of iNOS









inhibiton, we ran Western blots on NO and OL control plantaris to measure iNOS protein

levels.

Delimitations. Gender and species differences may exist in the hypertrophic

response to the functional overload model. We have chosen to study female

Sprague-Dawley rats.

Assumptions. Since the animals were housed in pairs, water consumption was

assumed to be equal between cage mates. It is also assumed that the animal did not favor

the contralateral limb during the OL period. Our lab previously showed that functional

overload induced by synergist ablation was a sufficient stimulus to show significant

differences between the overloaded plantaris and the contralateral control.

Significance of the Study

Attenuating muscle wasting is a pertinent clinical goal in combating pathologies

and conditions that lead to loss of function and impairment of ambulation. Aging and

disease lead to impaired skeletal muscle regrowth and regeneration, resulting in loss of

muscle mass and strength. Recovery from neurodegenerative disorders, spinal cord

lesions, and muscle unloading due to spaceflight, injury, or extended bed rest will be

more rapid if therapeutic strategies are discovered and implemented. Therefore, it is

critical to determine the signaling mechanisms responsible for the load-induced

phenotypic adaptations in skeletal muscle. Elucidating the biochemical pathways

controlling muscle growth and the phenotypic changes that support recovery from

injurious assault can lead to therapeutic strategies that prevent or possibly reverse muscle

atrophy. Progression in this basic science may aid in identifying targets for anti-atrophy

drugs.














CHAPTER 2
LITERATURE REVIEW

Adult skeletal muscle is highly responsive to changes in functional demands and,

therefore, serves as an excellent model in elucidating the molecules that serve as links

between mechanical stimuli and gene expression. Researchers employ clinically relevant

in vivo models as well as in vitro models due to the ease with which the mechanical

effects can be controlled and isolated. In whole animals, chronic overload causes

structural and phenotypic alterations in muscle fibers, resulting in dramatic muscle

growth. Likewise, myotubes subjected to stretch experience growth, proliferation and

associated fiber type changes in culture. The pathways governing these changes,

however, are poorly understood. Nitric oxide synthase (NOS) activity may be essential

during increased contractile activity, suggesting nitric oxide (NO) may act as a

coordinating master signal during muscle growth. The purpose of this study was to

determine if NOS activity inhibition would attenuate the increased expression of muscle

growth/regulatory factors and proteins associated with the contractile apparatus during

overload-induced hypertrophy. This chapter provides a critical review of the scientific

literature related to the proposed project along with interpretations where possible.

Skeletal Muscle Adaptations to Load

Adult skeletal muscle is extremely adaptable to changes in mechanical loading.

The adaptations to chronic overload involve many coordinated steps that lead to

structural and phenotypic changes within the muscle fibers. The resulting growth

involves the activation and fusion of satellite cells, the increased synthesis of structural









and contractile proteins, and subsequent capillary angiogenesis. Each of these steps is

controlled by specific growth and/or regulatory factors. Hepatocyte growth factor (HGF)

initiates satellite cell activation, insulin-like growth factor-1 (IGF-1) governs cell

proliferation and fusion, and activates protein synthesis, vascular endothelial growth

factor (VEGF) regulates angiogenesis, and myogenin (MGN) is involved in

differentiation and expression of the slow phenotype. Each of these factors and its

involvement in skeletal muscle growth following bouts of overload and/or stretch is

described below.

Hypertrophy and Fiber Type Shifts with Loading

Growth of skeletal muscle fibers in response to an increased mechanical load is a

complex event marked by large-scale remodeling of fiber architecture. The process is

associated with injury, immune cell infiltration, and regeneration (69), and involves the

activation and subsequent fusion of satellite cells to the muscle fibers (48, 57, 58), and

increased synthesis of structural and contractile proteins (28, 76). Increased protein

synthesis causes the accumulation of contractile and structural proteins resulting in the

addition of myofibrils and an accompanying increase in fiber cross-sectional area. The

increase in fiber cross-sectional area reflects an increase in muscle fiber diameter without

an increase in the number of muscle fibers (26, 72). Finally, the fiber type changes

associated with increased muscle recruitment are mediated via calcium-dependent

signaling, including calcium-calmodulin-dependent protein kinase and calcineurin. These

pathways interact with others to transcriptionally regulate expression of the myogenic

regulatory factor, myogenin (77). Myogenin, in turn, plays a major role in activation of

the myogenic program in differentiating satellite cells, as well as regulating the

slow-oxidative phenotype in adult myofibers (34).









Skeletal Muscle Hypertrophy and Insulin-Like Growth Factor (IGF-1) Signaling

IGF-1 signaling cascade. IGF-1 is an important regulator molecule in muscle

physiology, particularly important for maintenance and growth of adult skeletal muscle

mass (3). Although the major part of the circulating fraction of total IGF-1 is produced in

the liver, IGF-1 is also locally expressed in the muscle (1). This endogenous IGF-1 acts

in a paracrine/autocrine manner by binding to skeletal muscle IGF-1 receptor (IGFR).

IGF-1 binding to IGFR, leads to activation of the insulin receptor tyrosine kinase and

subsequent activation of several cytosolic substrates, such as calcineurin, the

mitogen-activated protein kinase/extracellular signal-related protein kinase

(MAPK/ERK) and the phosphoinositide 3'-kinase (PI3K). Interestingly, these pathways

have distinctive and possibly opposing end results. Calcineurin is a phosphatase that

targets a family of transcription factors, the nuclear factors of activated T-cells (NFAT).

Once dephosphorylated, cytosolic NFAT translocates to the nucleus to increase the

transcription of IGF-1-sensitive genes, specifically Type I (slow) myosin heavy chain

(MHC). Interestingly, IGF-1 also activates MAPK/ERK, a powerful myoblast

proliferative cascade that inhibits differentiation, and PI3K, whose activity ultimately

leads to increased protein synthesis and differentiation. However, IGF-1 is a more

powerful and potent activator of PI3K. Once activated, PI3K phosphorylates Akt/PKB

and initiates a protein kinase cascade that includes mammalian target of rapamycin

(mTOR), p70s6 kinase and increased protein synthesis. As a result, IGF-1 plays a major

role in the growth and maintenance of adult skeletal muscle and is a potent stimulator of

anabolic, mitogenic, and myogenic processes in skeletal muscle (51, 74).

The IGF-1 response in functionally overloaded skeletal muscle. While

circulating IGF-1 is important to these skeletal muscle responses, it is clear that the









autocrine/paracrine actions of endogenously produced IGF-1 are critical for muscle

hypertrophy (1). Perhaps the most convincing evidence that IGF-1 mediates adult skeletal

muscle hypertrophy comes from muscle-specific overexpression of IGF-1 in a transgenic

mouse model (15), and local infusion of IGF-1 to the rat tibialis anterior muscle (3). In

the first study, Criswell and colleagues discovered that the mice overexpressing IGF-1

demonstrated a higher absolute muscle mass in both the gastrocnemius and the tibialis

anterior (TA) compared to their wild-type counterparts. In the second study, the authors

implicated IGF-1 in acting directly to stimulate protein synthesis and satellite cell

proliferation, resulting in skeletal muscle hypertrophy. Further, these conclusions were

supported by the observed increase in absolute weight of the TA injected with IGF-1.

Additionally, the total protein and DNA content of these muscles increased. Both of

these models produced dramatic muscle hypertrophy without any change in loading

pattern, indicating the sufficiency of IGF-1 for skeletal muscle hypertrophy.

Not only is IGF-1 necessary for skeletal muscle growth, its expression is increased

during periods of increased load. Functionally overloaded rat plantaris muscles

experienced a significant increase in IGF-1 mRNA as well as the compensatory increases

in muscle size and fiber cross-sectional area (CSA) when compared to the contralateral

control plantaris (78). Interestingly, these rats were hypophysectomized to decrease the

levels of pituitary-derived growth hormone, illustrating the inherent importance of IGF-

l's autocrine and paracrine properties to muscles during overload-induced hypertrophy.

Regenerating skeletal muscle also shows an increase in the IGF-1 signal. Rat TA

muscles recovering from bupivicaine injection demonstrated temporal and maximal

expressions of IGF-1 mRNA at days 5 and 10, respectively (43). Further, Yang and









colleagues (79) showed that IGF-1 is strongly correlated with the phenotypic adaptations

associated with stretch and overload. Therefore, IGF-1 signaling is important for both

skeletal muscle growth and the phenotypic changes associated with this growth. Adams

and Haddad (2) suggested that the increases in muscle IGF-1 preceded the phenotypic

changes in the functionally overloaded rat plantaris. These IGF-1 mRNA increases

peaked at 3 days of overload with a maximal increase of 4 times normal levels. Finally,

McCall and colleagues (44) showed that IGF-1 is transcriptionally regulated in vivo

during hypertrophy as evidenced by the induction of the endogenous IGF-1 pre-mRNA

during functional overload of the rat plantaris.

IGF-1 induction in cell culture. In vitro studies have also been used to elucidate

the pathways that participate in and activate muscle growth and the phenotypic changes

associated with the growth. The research designs most commonly used are myotubes

subjected to stretch or electrical stimulation. Media depleted of mitogens is commonly

used to activate differentiation, as it induces the expression of localized IGF-1 (83). This

induction overrides the negative control of serum mitogens on differentiation. Electrical

stimulation (ES) produces changes in metabolic enzyme and contractile protein gene

expression, and stretch potentiates the ES-induced changes, including harvesting a more

robust IGF-1 response (46). Bayol et al.(6), suggest that proteins associated with the

IGF-1 axis, in part, mediate these changes. Addition of IGF-1 to culture media stimulates

both MAPK/ERK and PI3K/Akt signaling pathways during differentiation, but not to the

same degree, suggesting the necessity of both pathways during proliferation and the

induction of differentiation (22, 63). IGF-1 enhanced in vitro muscle growth through

increased satellite cell proliferation via the PI3K/Akt pathway (12). Finally, repetitive









stretch/relaxation of differentiated muscle cells stimulated the acute release of IGF-1

during the first 4 hours of stretch (51). In vivo and in vitro models suggest that IGF-1

uniquely stimulates both muscle cell proliferation and differentiation.

MGF acts as a mechanically sensitive splice variant. Although IGF-1 is

essential to muscle growth and maintenance, recent evidence suggests a splice variant of

the growth factor that may be particularly responsive to loading and/or injury. Goldspink

and colleagues (81) have identified and cloned this splice variant of IGF-1, called

mechano growth factor (MGF). MGF, produced by active muscle, controls local tissue

repair, maintenance, and remodeling through induction of local protein synthesis, and

prevention of apoptosis. The alternative splicing of the IGF-1 mRNA results in a 52 base

pair insert in the E domain that likely causes it to bind to a different protein. The reading

frame shift associated with the MGF peptide also causes it to have a different C terminal

sequence, inducing differential protein/receptor affinities. Therefore, the MGF peptide

may mediate slightly different processes. In vitro, MGF inhibits terminal differentiation

of C2C12 cells and increases cell proliferation by processes that are independent of the

IGF-1 receptor (82). MGF expression increases with high resistance exercise in humans

(30), following acute local damage in rat muscle (32), and with in vivo electrical

stimulation and stretch (13). The peak in MGF expression is approximately on the third

day of differentiation. Therefore, local production and post-transcriptional processing of

IGF-1 is critical for adaptations in muscle mass in response to altered loading.

Nevertheless, the signals) linking mechanical load and skeletal myofiber IGF-1 gene

expression remain a mystery.









Skeletal Muscle Hypertrophy and Vascular Endothelial Growth Factor (VEGF)

Skeletal muscle hypertrophy is temporally coupled to the induction of angiogenesis

and is closely linked to blood vessel recruitment and capillary angiogenesis, such that

capillary density is maintained during the early stages of hypertrophy (54). Regular

contractile activity induces an increase in capillarization. Along with basic fibroblast

growth factor (bFGF), VEGF regulates angiogenesis, and is highly induced in growing

and hypertrophying skeletal muscle (66). VEGF is a potent mitogen of endothelial cells.

VEGF binds to two primary receptors on endothelial cells (VEGFR1 and VEGFR2). In

addition to inducing endothelial cell proliferation, migration and differentiation,

activation of VEGFR2 leads to NO production via eNOS. NO production can be vital to

VEGF signaling, as inhibiting NO production diminishes the angiogenic response (7, 41).

Recently, expression of VEGF in skeletal muscle cells has been shown to be under the

control of IGF-1-dependent Akt signaling (66) and its expression is increased via an

AMPK-p38 MAPK signaling cascade.

VEGF expression and skeletal muscle overload. Increased angiogenesis is

necessary to support a growing muscle as its metabolic demands increase. In humans

(56), dogs (31), and rats (80), increased metabolic demands associated with an increased

exercise paradigm caused enhanced remodeling of capillaries local to the muscle, and

increased muscle contraction in rats caused an upregulation of VEGF (4). Although

many models have induced angiogenesis by way of increases in blood flow and

accompanying capillary shear stress, few have investigated the isolated role of muscle

contraction and overload on growth factor expression and release, and on endothelial cell

proliferation.









Skeletal muscle overload is a potent stimulus for capillary growth in vivo. Rat

extensor digitorum longus muscles exhibited an increase in capillary-to-fiber ratio after

removal of the synergist tibialis anterior muscle (19), thereby implicating the importance

of capillary angiogenesis during synergist ablation and the resulting overload. Degens

and colleagues (17) found that VEGF mRNA expression increased after 2 weeks of

compensatory overload following functional elimination of synergists to the plantaris,

and this expression precedes capillary proliferation. This timepoint coincides with the

half time for increases in capillary supply, suggesting VEGF plays a preparatory role in

capillary angiogenesis and may be elevated during the week of recovery post surgery.

Additonally, NO donors increase VEGF mRNA in vivo. Benoit and colleagues (7) found

that injecting rats with nitroprusside, an NO donor, resulted in elevated levels of the

growth factor in the gastrocnemius muscle. Further, another NO donor,

S-nitroso-N-acetylpenicillamine (SNAP) induced angiogenesis in vivo and in vitro (41).

Endothelial cells treated with SNAP demonstrated increased cell migration and

differentiation into capillaries. Therefore, there is strong evidence that NO acts as a

potential coordinator of capillary angiogenesis via transcriptional regulation of VEGF.

VEGF and skeletal muscle cells. In addition to playing an important role in

capillary angiogenesis, VEGF has also been implicated in stimulating skeletal muscle

fiber regeneration in vivo. Arsic and colleagues (5) delivered VEGF using an

adeno-associated virus vector to animals subjected to unilateral ischemia and muscle

injury. As a result, VEGF delivery significantly reduced the damaged area and increased

the number of regenerating fibers when compared to control muscle. The same

investigators also found that VEGF increased the number, length, and nuclear content of









differentiated C2C12 cells. VEGF decreased the rate of proliferation and promoted

differentiation, a result that paralleled VEGF's ability to protect the differentiating cells

from apoptotic death. Taken together, these data suggest the importance of investigating

VEGF as a target for a possible therapeutic role in muscular disorders.

VEGF receptors are members of a family of tyrosine kinase receptors that are

expressed by a variety of tissues. Initially described on endothelial cells, recent data has

shown the presence of VEGF receptors on hematopoietic and vascular smooth muscle

cells. More important to the present study, others present evidence of VEGF receptor

expression on cultured myoblasts and myotubes, and on skeletal myofibers isolated in

culture. This helps to explain the close association of myocyte remodeling with

angiogenesis.

Hepatocyte Growth Factor (HGF) and Muscle Growth

Satellite cells are skeletal muscle progenitor cells. Skeletal muscle satellite cells

are myogenic stem cells residing between the sarcolemma and the basement membrane in

postnatal skeletal muscle. Normally quiescent, they are activated in response to

mechanical changes in the muscle. They actively participate in muscle regeneration

following injury and are required for load-induced hypertrophy. Inactivation of satellite

cells by gamma irradiation prevents muscle hypertrophy following compensatory

overload. Once activated, the satellite cells migrate to the area of injury or growth to

supply myonuclear precursors to the expanding fibers, preserving the myonuclear domain

inherent to adult skeletal muscle. In response to stretch or compensatory overload,

satellite cells are activated, enter the cell cycle and subsequently proliferate. Schiaffino

and colleagues (61) have shown that this proliferative activity begins at -3 days after









compensatory overload. Activated satellite cells fuse into preexisting fibers or form new

fibers (45).

Although other growth factors have the capacity to stimulate satellite cell

proliferation and/or differentiation (IGFs and fibroblast growth factors (FGFs)), it

appears that hepatocyte growth factor (HGF) initiates satellite cell activation. It is active

as a heterodimer that results from proteolytic cleavage of the inactive, single chain pro-

form. Present in the liver, kidney, lung, and spleen, HGF message and protein have also

been localized in adult, uninjured skeletal muscle sequestered to the extracellular matrix

(62, 67). The signaling receptor for HGF is the c-met receptor, and both its message and

protein have been found in satellite cells in vitro and in vivo. Muscle stretch or injury

releases HGF from the extracellular matrix to bind the c-met receptor, thereby inducing

the satellite cells to enter the cell cycle and proliferate. Cultured satellite cells also

express HGF, which acts in an autocrine fashion to induce proliferation (62).

Additionally, chronic low frequency stimulation of rat muscle in vivo leads to the

activation and proliferation of satellite cells. Further, this increase in satellite cell

progeny paralleled the increase in myonuclear content (55). However, the link between

changes in external mechanical and electrical stimuli and chemical signaling is poorly

understood.

HGF release and compensatory overload. Overloaded plantaris muscle shows an

increase in HGF message. After unilateral removal of the synergists to the plantaris,

HGF mRNA expression increased at 3, 7 and 21 days when compared to the contralateral

control (78). Plantaris hypertrophy was closely associated with the upregulation of HGF

and FGF mRNA, even when the plantaris muscles were denervated during the overload









period. This suggests that HGF expression may be involved in overload-induced

hypertrophy.

HGF and stretch. HGF release from the extracellular space and subsequent

satellite cell activation appears to depend on external load. Cultured quiescent satellite

cells subjected to stretch entered the cell cycle earlier than those in control conditions,

and conditioned media from these stretched cells activated unstretched satellite cells (68).

Additionally, blocking HGF in stretched cultures prevented satellite cell activation. In

vitro experiments suggest that acute satellite cell activation is mediated by release of

local HGF already present in skeletal muscle. NO serves as the link between mechanical

stretch in vitro and HGF release from satellite cells. Tatsumi et al. (67) found that NOS

activity is increased in cyclically stretched cells and the resulting NO mediates HGF

release.

HGF is synthesized and secreted by satellite cells in vitro, suggesting HGF

possesses both autocrine and paracrine properties (62). Further, it is suggested that

isolated satellite cells also express and secrete an HGF activator to ensure the active form

of HGF will be available for autocrine action. Other progenitor cells, specifically

fibroblasts, do not express HGF message or protein, lending support to the idea that

satellite cells are the cells responsible for increasing HGF levels upon activation. Based

on these data, it seems likely that sustained hypertrophic activity may require

upregulation of HGF expression in activated satellite cells and/or muscle fibers

Myogenin (MGN) and Control of Adult Muscle Phenotype

The myogenic regulatory factors (MRFs), including MGN, MyoD, myf-5, and

MRF-4, belong to the basic helix-loop-helix superfamily of transcription factors. These

proteins form heterodimers with ubiquitous E-proteins and bind to DNA at consensus E-









box domains, present in many muscle-specific gene promoters and drives the

differentiation process (52). During muscle development, the MRFs are expressed in a

highly regulated temporal pattern, with MGN appearing late in differentiation

corresponding to the beginning of myoblast fusion.

MGN continues to be expressed in adult skeletal muscle fibers and is believed to

control the slow-twitch fiber phenotype. MGN is an important regulatory factor during

overload-induced hypertrophy. Its expression in activated satellite cells corresponds to

terminal differentiation of these cells and fusion with existing muscle fibers, a process

that is required for normal hypertrophy of adult muscle fibers.

Hypertrophy and Nitric Oxide

The exquisite coordination of the complex response to skeletal muscle overload

suggests a common regulator. Smith et al. (64) reported that inhibition of nitric oxide

synthase (NOS) activity prevents the normal hypertrophy and fiber type adaptations to

chronic skeletal muscle overload. Further, the release of HGF during skeletal muscle

loading is nitric oxide synthase-dependent (68), and endurance exercise increases VEGF

expression in skeletal muscle via a nitric oxide-dependent mechanism (24). Together,

these data suggest that nitric oxide may be acting as a common master signal. Therefore,

we hypothesized that inhibition of NOS activity would prevent the increased local

expression of growth/regulatory factor mRNAs (IGF-1, MGF, HGF, VEGF, and

myogenin) and Type I (slow) myosin heavy chain (MHC) expected during the early

stages of functional overload in the rat plantaris.

NOS activity and fiber type composition. Adult skeletal muscle is a mosaic of

four different fiber types, one slow (I), and three fast (IIa, IIb, IId/x). These differ with

respect to specific biochemical and morphological characteristics. Skeletal muscle









possesses a high degree of plasticity, allowing it to respond and adapt to altered

physiological demands by switching between fiber types. The adaptation to functional

demands is a well-documented process, known as fast-to-slow or slow-to-fast

transformation. A fast-to-slow transformation is induced by muscle growth

(hypertrophy), and results in an increased oxidative capacity and a greater resistance to

fatigue. Conversely, a slow-to-fast fiber transformation characterizes muscle wasting

(atrophy) and heightens a muscle's fatigability and glycolytic capacity. Expression of the

different fiber isoforms is determined by the type of nerve (8), the level of physical

activity (60), and the amount of passive stretch (59). There has been extensive research

in uncovering the pathways or molecules that are responsible for directing the fiber type

transition, including NO.

The metabolic phenotype of skeletal muscle fibers, including fiber type, is

controlled primarily by calcium-dependent signaling. The tonic activity of slow motor

neurons produces a chronic low-level of cytoplasmic calcium in slow-twitch fibers. This

activates calcium-dependent signals. Most notably, the calcium-dependent phosphatase,

calcineurin, and the calcium-calmodulin-dependent protein kinases (CaMK). The

constitutive NOS enzymes are also calcium-calmodulin dependent and may play a

regulatory or facilitative role in the control of muscle fiber type. Smith and colleagues

(64) demonstrated the involvement of NOS in skeletal muscle adaptations to overload.

As in previous research, overload induced hypertrophy and a fast-to-slow fiber type shift.

Treatment with the NOS inhibitor, L-NAME, however, attenuated the overload-induced

hypertrophy and prevented the increase in type I fibers with hypertrophy. This suggests

that NO is necessary for changes in skeletal muscle phenotype in vivo. Additionally, NO









covalently modifies thiol groups on the ryanodine receptors associated with calcium

channels of the sarcoplasmic reticulum. This modification results in a release of calcium

into the cytosol (21). Therefore, NO could facilitate activation of calcium-dependent

pathways. Planitzer et al. (53) discovered that fast-oxidative fibers carry the highest

concentration of NO S-1, possibly facilitating the fiber type transitions through a slower

phenotype. Future research on NOS activation and the associated changes in phenotype

is important in elucidating the complex signaling pathways associated with hypertrophy

and muscle fiber type.

Summary

Disease states and conditions such as spaceflight and extended bed rest unload

skeletal muscle and cause muscle atrophy. The characteristic phenotypic changes

associated with muscle wasting are a serious clinical concern and modalities to prevent

these changes should be investigated. Defining the mechanisms by which intracellular

and extracellular signaling molecules control skeletal muscle growth shows promise for

developing novel therapeutics to combat muscle wasting. The rat model of unilateral

synergist removal provides a robust hypertrophic response, making this model a valuable

tool in elucidating the regulatory pathways. Nitric oxide is a ubiquitous signaling

molecule, located in essentially every tissue in the body. Inhibition of nitric oxide

synthase (NOS) activity interferes with the muscle growth response. However, NO's

specific role in muscle hypertrophy remains a mystery. Previous research implicates NO

signaling in the fiber type transition associated with muscle growth. Further, NO plays a

role in satellite cell activation and proliferation. Therefore, NO may act as a coordinating

molecule in the many different growth steps of skeletal muscle. This project will






22


determine whether blocking NOS activity can attenuate the expression of various

growth/regulatory factors during functional overload of the plantaris muscle.















CHAPTER 3
METHODS


L-NAME & TRIM


Sacrifice & Tissue Harvesting


Day -2 Day 0 Day 5

Figure 3-1. Experimental design flowchart. Animals were given the drugs starting 2 days
before the surgeries. Tissue extraction was on day 5.

Animals

The subjects were adult (-4 month-old) female Sprague-Dawley rats (-250 g). All

were housed in the J. Hillis Miller Animal Science Center and fed the same diet (rat chow

and water ad libitum) throughout the experiment. They were maintained on a 12 h

light:dark photoperiod (light 0700 to 1900h). All procedures followed NIH guidelines









and were approved by the University of Florida's Institutional Animal Care and Use

Committee.


Inhibition of NOS Activity

After an acclimation period of one week after arrival, the rats were randomly

divided among the three experimental groups. The pharmacological inhibition of nitric

oxide synthesis was achieved by administering the competitive non-isoform-specific

NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical) or the

nNOS-selective inhibitor, 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM, Cayman

Chemical) during the treatment period. L-NAME (Img/ml) was added to the drinking

water to maintain a dose of -90 mg/kg/d. TRIM was dissolved in phosphate buffered

saline (PBS) and injected intraperitoneally (IP) at a concentration of 10 mg/kg/d. To

control for the possible confounding influences of a daily IP injection, the Control and

L-NAME animals were injected daily with a volume of PBS equal to the volume injected

into the TRIM animals.

Synergist Ablation Surgery

Chronic overload of the plantaris was induced by surgical, unilateral removal of the

synergist muscles to the plantaris. The rats were anaesthetized with inhaled isoflurane

(2-5%) with oxygen as the carrier gas. Using aseptic technique, a midline incision was

made in the skin of the right hind limb, from the popliteal fossa to the Achilles tendon

region. A second longitudinal incision was made through the hamstrings exposing the

distal gastrocnemius and Achilles tendon region. The gastrocnemius tendon was carefully

separated from the plantaris tendon, and the gastrocnemius muscle sectioned. The distal

two-thirds of the gastrocnemius was removed, taking care not to disturb the plantaris









nerve and blood supply. Next, the soleus muscle was carefully isolated and removed. The

hamstring incision was closed with 4-0 vicryl absorbable suture. The overlying skin was

closed with sterilized metal wound clips and treated with a topical antibiotic cream to

avoid infection.

A sham operation was performed on the left leg of each rat. In this procedure, the

same incisions were made as above and the gastrocnemius tendons were isolated without

transecting the muscles. The animals were allowed to fully recover from the anesthetic

before returning to their cages. The rats were examined daily for signs of infections or

wound openings, which were promptly treated, if found.

Experimental Protocol

Two days before the surgery, L-NAME was added to the drinking water of the

appropriate group, and daily injections of TRIM (TRIM group) or PBS (Control and

L-NAME groups) were begun. Forty-eight hours after the start of drug treatments, the

animals underwent the ablation surgeries. All animals received the unilateral synergist

ablation surgery along with the sham surgery on the contralateral limb. After a brief

recovery period, the rats were group-matched, housed in pairs, and allowed to ambulate

freely. Water was replaced each day, and body mass and water consumption were

recorded daily throughout the experimental period. The dose of L-NAME was calculated

for each rat. On day 5 post surgery, the animals were anesthetized with inhaled

isoflurane and sacrificed by exsanguination. The plantaris muscles were immediately and

bilaterally removed, trimmed of excess connective tissue and fat, weighed on an

analytical scale, and flash-frozen in liquid nitrogen. Frozen muscles were powdered

using a liquid nitrogen-cooled mortar and pestle. The powdered muscle was divided into

separate tubes and stored at -800 C for subsequent biochemical analyses.









Nitric Oxide Production

To determine the efficacy of the pharmaceutical NOS blockers, L-NAME and

TRIM, serum nitrate/nitrite levels were measured. Whole blood (- 4 mL) collected from

the animals at the time of sacrifice was incubated for 30 minutes at room temperature to

allow for clotting. The samples were centrifuged 20 minutes at 5000g to separate the

serum from the cellular fraction. The serum was removed and stored at -800C until

further analysis. Using a pre-designed kit (Cayman Chemical), the serum samples were

analyzed for nitrate/nitrite levels as per the manufacturer's specifications.

Reverse Transcription and Real-Time Quantitative PCR

Total RNA was isolated using Trizol Reagent (Life Technologies, Carlsbad, CA)

according to the manufacturer's instructions. The amount of total RNA was evaluated

by spectrophotometry and the integrity checked by gel electrophoresis. Total RNA (5

[tg) was reverse transcribed using the Superscript III First-Strand Synthesis System (Life

technologies, Carlsbad, CA) using oligo(dT)20 primers and the protocol outlined by the

manufacturer. One ptL of cDNA (5 ptL for MGF) was added to a 25 ptL PCR reaction for

real-time PCR using Taqman chemistry and the ABI Prism 7000 Sequence Detection

System (Applied Biosystems (ABI), Foster City, CA). The comparative Ct method (ABI

User Bulletin #2) was employed for the relative quantitation of gene expression.

Hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as the normalizer.

The enzyme is important in purine biosynthesis, and it was chosen based on initial

experiments showing that our manipulations did not affect the expression of the

transcript. Five-fold dilution curves were assayed on selected samples to confirm the

validity of this quantitation method for each gene. IGF-1(GenBank NM_178866), HGF









(GenBank NM_017017), skeletal a-actin, Type I (slow) MHC, and myogenin (GenBank

NM_017115) mRNA transcripts were assayed using pre-designed rat primer and probe

sequences commercially available from Applied Biosystems (Assays-on-Demand). MGF

(5'-CACTGACATGCCCAAGACTCA (forward) and

5'-CTTTGCAGCTTCCTTTTCTTGTG (reverse)) and HPRT

(5'-GTTGGATACAGGCCAGACTTTGT (forward) and

5'-AGTCAAGGGCATATCCAACAACAA (reverse)) mRNA were assayed using

custom made primers (Applied Biosystems, Assays-by-Design). The MGF reverse primer

was custom designed to span the 52-base pair insert unique to the rat MGF cDNA.

Semi-Quantitiave RT-PCR

Because the pre-designed rat primers and probes supplied by ABI did not

differentiate among the four isoforms of VEGF, we chose to do semi-quantitative PCR to

determine if there was differential expression between the isoforms associated with this

model. Total RNA was isolated as described above and reverse transcribed using Ready-

to-Go You-Prime First-Strand Beads (Amersham Biosciences, Buckinghamshire, UK)

and a combination of random hexamers and oligo(dT)20 primers. cDNA for the four

isoforms of VEGF expressed in rat skeletal muscle were amplified simultaneously using

published primer sequences and conditions (16), with expected sizes of 632-, 560-, 500-,

and 428-bp PCR products for amplification of VEGF-188, VEGF-164, VEGF-144 and

VEGF-120, respectively. 18S ribosomal cDNA was amplified simultaneously as an

internal control for amplification efficiency (Ambion, QuantumRNA internal standards).

PCR products were separated by electrophoresis on 1% agarose gels, stained with

ethidium bromide, and band intensity was quantified by densitometry (Scion Image

software).









Immunoblotting

It has been shown that TRIM has significant inhibitory effects on both nNOS and

the inducible form ofNOS, iNOS. Both nNOS and eNOS are constitutively active in

skeletal muscle, while iNOS activity is normally absent. However, iNOS protein

expression and activity can be induced in skeletal muscle by the inflammatory process.

To assess the possibility of the potentiation effects of iNOS, we evaluated iNOS protein

levels via Western blotting in the normal loaded (NL) and the overloaded (OL) plantaris

muscles from the Control group. For all Western blots, the powdered muscle was

homogenized in 20 mM Tris (pH 7.5), 150 mM NaC1, 1 mM EDTA, 1 mM EGTA, 1%

Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM 3-glycerol phosphate, 1 mM

sodium orthovanadate, 1 [tg/ml leupeptin, 1 mM PMSF, and 10 [tg/ml aprotinin using a

stainless steel blade. Homogenates were centrifuged for 10 min at 1000g to remove

connective tissue and cellular debris and protein content quantified in the supernatant

using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA, USA).

For iNOS protein, 80 [tg of total protein were subjected to SDS-PAGE on 7%

polyacrylamide gels. Separated proteins were transferred to polyvinylidene difluoride

(PVDF) membranes under cold conditions, which were stained with Ponceau S (0.1%

w/v in 5% acetic acid) to verify equal loading. The membranes were subsequently

blocked for Ih at room temperature in Tris-buffered saline-Tween (TBST; Tris-HCl (pH

7.4), 150 mM NaC1, 0.05% Tween 20) containing 5% nonfat dry milk and then incubated

(40C overnight) with primary antibody for iNOS (Transduction Laboratories) diluted in

blocking solution. The membranes were washed three times in TBST, 10 minutes each,

followed by treatment with horseradish peroxidase-labeled anti-mouse antibody (Vector









Labs) for 2h at room temperature. Reactions were developed using the Enhanced

Chemiluminescence detection reagents (ECL; Amersham Biosciences, Buckinghamshire,

UK) according to the manufacturer's instructions, and protein levels were determined by

densitometry (Scion Image software).

For p70S6K, membranes were then blocked for 1 h in Tris-buffered saline-Tween

(TBST; Tris-HCl (pH 7.4), 150 mM NaC1, 0.05% Tween 20) containing 5% nonfat dry

milk and incubated with primary antibody for phosphor(Thr389)-p70S6K (Cell Signaling

Technology, Beverly, MA) overnight at 40C. Membranes were washed three times in

TBST, 10 min each, followed by incubation with a peroxidase-labeled anti-rabbit

antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at RT. Reactions

were developed using the Enhanced Chemiluminescence detection reagents (ECL;

Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's

instructions, and protein levels were determined by densitometry (Scion Image software).

Limitations

Although our lab has had previous success in using the synergist ablation model to

induce overload hypertrophy, the procedure is not without criticism. The invasive nature

of the surgery could affect skeletal muscle gene expression independent of mechanical

overload. Nevertheless, comparison of overloaded muscles to contralateral normally-

loaded muscles (i.e. within-subject design) controls for potential effects of systemic

factors, such as circulating cytokines, hormones, or inflammatory cells. Further, sham

surgeries control for local surgical effects. It is impossible to determine the source of

intramuscular NO in the present design. Future studies will be necessary to examine

potential intrinsic and extrinsic sources of NO signaling during muscle overload.









Our model investigated the effects of 5 days of overload on gene expression of

several growth factors, myogenin, and contractile proteins. This time point was chosen

based on published accounts of overload-induced IGF-1 and MGN expression, protein

synthesis, and RNA and DNA accumulation. All of these factors are significantly

elevated in the overloaded plantaris at 5 days. However, the dynamics associated with

HGF and VEGF induction is unknown, as is the time course of NO signaling. Therefore,

more studies utilizing the functional overload model at different time points during the

hypertrophic response may be necessary to elucidate possible differential gene

expression.

Vertebrate Animals

Female Sprague-Dawley rats were used in this research. This study required the

removal of hindlimb muscles synergistic to the plantaris via a non-terminal surgery, with

subsequent ambulation. This invasive procedure prevented the use of human subjects.

Sprague-Dawley rats were selected based on the large amount of preliminary data

collected with this model in our lab and many others.

Statistical Analysis

This experiment was designed to test the hypothesis that NOS activity is necessary

for the induction of growth factor and contractile protein expression during overload-

induced hypertrophy. A 3 x 2 (treatment x loading condition) ANOVA with repeated

measures on the loading condition was employed to determine main effects and

interactions for each variable. Where significant differences were found, Tukey's HSD

test was implemented post hoc to determine individual group differences. Significance

was established apriori at p<0.05.














CHAPTER 4
RESULTS

Systemic and Biological Responses to Treatment

Body mass did not change from pre- to post-overload treatment for any group.

Further, body mass did not differ between groups at any time point (Table 4-1).

Consistent with a report from Adams and Haddad (2), no OL-induced changes in total

protein were observed at 5 days of overload. Nevertheless, this model does cause muscle

protein accumulation following 14 days of OL (64). This study focuses on the early

signaling events leading to this protein accumulation. Water consumption in the L-

NAME-treated group was reduced -40% during the first 24h of L-NAME treatment, but

then returned to normal (not different from the Control group; data not shown) for the

remainder of the treatment period. The average L-NAME dose was 89.2 mg/kg/d.

L-NAME and TRIM treatments significantly lowered serum nitrate/nitrite levels

(P<0.05), indicating successful systemic NOS inhibition. Mean (SEM) serum

nitrate/nitrite levels were: Control = 7.080.31 tiM, L-NAME = 1.650.09 tiM, and

TRIM = 4.490.19 tM.

Myogenin mRNA Expression

Five days of plantaris overload caused an approximately 4-fold increase in

myogenin mRNA expression. No significant differences were observed between

treatment group (Table 4-2).









Contractile Protein mRNA Expression

Five days of OL resulted in a 90% increase in skeletal a-actin mRNA and a 140%

increase in type I (slow) MHC (MHC-I) mRNA (Figure 4-1; Control NL vs. Control OL).

However, these data indicate a complete inhibition of this OL-induced response in the

NOS-inhibited groups. Skeletal a-actin and MHC-I mRNA levels did not differ between

NO and OL muscle in either treatment group (L-NAME and TRIM). Further, skeletal a-

actin and MHC-I transcript levels in L-NAME and TRIM muscles did not differ from that

in control NL muscles.

Growth Factor mRNA Expression

Expression of mRNA for HGF, IGF-1, MGF, and the 120 amino acid splice variant

of VEGF (VEGF-120) were all increased in the OL muscles (Table 4-2). IGF-1 mRNA

was increased -4 fold and MGF mRNA -9 fold in Control/OL compared to Control/NL

muscles (Figure 4-2). TRIM treatment did not affect IGF-1 or MGF mRNA expression in

the NL muscle, but approximately doubled expression of both transcripts in the OL

muscle compared to Control/OL (Table 4-2). HGF and VEGF-120 mRNAs were

increased 15-20 fold in the OL muscles compared to NL, with no effect of either L-

NAME or TRIM treatment (Table 4-2 and Figure 4-3). Transcripts for VEGF-188,

VEGF-164, and VEGF-144 tended to be reduced in the OL muscles, but this did not

reach statistical significance (p>0.05). Real-time PCR assessment of total VEGF mRNA

showed a -50% reduction in OL muscle, compared to NL. The discrepancy between real-

time assessment of total VEGF mRNA (Table 4-2) and semi-quantitative assessment of

VEGF isoform expression (Figure 4-3) is most likely due to the relatively small

contribution of VEGF-120 to the total VEGF mRNA pool and the variability in the semi-









quantitative RT-PCR method, which failed to demonstrate a significant OL-related

reduction in the more abundant VEGF isoforms.

Phosphorylation of p70S6K

Expression of total p70s6k protein was increased in OL muscles in all three

treatment groups. Likewise, phosphorylated p70s6k was increased in OL muscles

compared to NL. The ratio of phosphorylated to total p70s6k did not differ between

Control NL and OL muscles. Conversely, the ratio of phospho to total p70s6k was

significantly elevated in OL muscles from L-NAME and TRIM animals, indicating a

greater relative phosphorylation status (Figure 4-4) in these muscles.

iNOS Protein Expression

TRIM significantly inhibits nNOS and, to a lesser degree, iNOS. Therefore, we

sought to determine if iNOS induction during overload could contribute to the observed

nitric oxide-dependent effects. iNOS protein was not detected in either normally loaded

or overloaded plantaris muscle (Figure 4-5), suggesting that the TRIM effects are due to

inhibition of nNOS activity.

Table 4-1. Body mass, plantaris mass, and total protein data for the overloaded rats.
Body Mass Plantaris mass Total Protein
(g) (mg) (mg/muscle)
Control 340.4 + 8.9 NL 323.0 + 21.1 38.8 3.8
OL 4,. S+ 13.9 36.1 4.0
L-NAME 334.4 + 11.5 NL 303.3 6.1 40.9 2.4
OL 387.0 + 22.1 43.5 3.5
TRIM 340.1 + 9.8 NL 342.5 19.3 36.5 3.7
OL 385.0 + 20.4 41.8 + 4.6
Definition of abbreviations: L-NAME = No-nitro-L-arginine methyl ester; TRIM = 1-(2-
trifluoromethyl-phenyl)-imidazole; OL = 5-day overloaded plantaris; NL = contralateral
normally loaded plantaris. Values represent means SEM.









Table 4-2. Real-time PCR quantification of mRNA transcripts for selected growth factors
and a regulatory gene in the plantaris muscle. Transcripts are normalized to
hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and
expressed relative to the Control, normally loaded (NL) value, using the
comparative Ct method.
Control L-NAME TRIM
NL OL NL OL NL OL
Transcript
MGN 1.38 + 5.28 + 1.09 + 3.58 + 0.89 + 3.68 +
0.32 0.81a 0.33 0.39ab 0.16 0.79ab
IGF-1 1.12 3.98 1.06 5.06 1.22 8.49
0.18 0.28a 0.18 0.59 ab 0.14 0.86' ab
MGF 1.07 8.84 0.73 11.32 0.99 22.03
0.33 0.78a 0.12 3.05 ab 0.29 6.31 a,'b
HGF 1.18 + 19.76 + 2.45 20.78 1.85 + 17.78
0.30 4.1 0.80 2.7 ,b 0.39 0.62 a,b
total 1.22 0.58 1.68 0.68 1.94 0.52
VEGF 0.27 0.09 0.52 0.14,b 0.50 0.08 ,b
Definition of abbreviations: L-NAME = N-nitro-L-arginine methyl ester; TRIM = 1-(2-
trifluoromethyl-phenyl)-imidazole; OL = 5-day overloaded plantaris; NL = contralateral
normally loaded plantaris; MGN = myogenin; IGF-1 = insulin-like growth factor-1; MGF
= mechanosensitive growth factor; HGF = hepatocyte growth factor; VEGF = vascular
endothelial growth factor. Values represent means SEM.
a = Significantly different from Control NL, p<0.05.
b = Significantly different from corresponding NL (within group), p<0.05.
c = Significantly different from Control OL, p<0.05.












2.5 A
a
SNL
2.0 -OL

a1.5
<
S1.0 -




0.0
Control L-NAME TRIM


B
3.5
a
3.0
00 ONL
2.5 H OL

2.0

1.5

S1.0

.0.5

0.0
Control L-NAME TRIM

Figure 4-1. Real Time PCR assessment and quantification of contractile protein mRNA
transcripts. A) skeletal a-actin mRNA level relative to HPRT mRNA in 5-day
overloaded (OL) and contralateral normally loaded (NL) plantaris muscles of
Control, L-NAME and TRIM-treated rats. B) Type I (slow) myosin heavy
chain (MHC) mRNA level relative to HPRT mRNA in 5-day overloaded (OL)
and contralateral normally loaded (NL) plantaris muscles of Control, L-
NAME and TRIM-treated rats. Values represent means + SEM.a =
Significantly different from corresponding NL, p<0.05.











121 A
O NL
SNL a,b
10 flOL



6 a











O NL
a
4





2

S20




Control L-NAME TRIM
30 a,b
QNL
25 OL














Figure 4-2. Real Time PCR quantification of insulin-like growth factor mRNA
a

a


5 1


Control L-NAME TRIM



Figure 4-2. Real Time PCR quantification of insulin-like growth factor mRNA
transcripts. A) Insulin-like growth factor-1 (IGF-1) mRNA level relative to
HPRT mRNA. B) Mechano growth factor (MGF) mRNA level relative to
HPRT mRNA. Values represent means + SEM. a = Significantly different
from Control/NL, p<0.05. b = Significantly different from Control/OL,
p<0.05.











DNL A
25- OL
< a
Sg20I
o a I



10'




0
'1 >




Control L-NAME TRIM

Control L-NAME TRIM
NL OL NL OL NL OL B
VEGF88s
VEGF164 -1
VEGF144-
VEGF120-


18S W -



Figure 4-3. Semi-quantitative RT-PCR analysis of VEGF mRNA splice variant
expression. A) Expression levels of VEGF-120 mRNA, relative to ribosomal
18S RNA in 5-day overloaded (OL) and contralateral normally loaded (NL)
plantaris muscles of Control, L-NAME and TRIM-treated rats. B)
Representative ethidium bromide stained 1% agarose gel illustrating PCR
products following amplification of VEGF and 18S. See Methods for details
of assay conditions. Values represent means + SEM. a = Significantly
different from Control/NL, p<0.05.











Control L-NAME TRIM
MW Markers
NL OL NL OL NL OL
Phos-p70s6k >g 1580 kDa


Total-p7Os6k >o pI 80 kDa
S50 kDa


Beta-actin >


P Q IP 39 kDa


10.0


6.0

4.0

2.0

0.0


O-NL
*OL


Control


L-NAME TRIM


Figure 4-4. Western blot analysis of p70s6K. A) Representative immunoblot for
phosphor(Thr389)-p70s6K, Total- p706K, and beta-actin (loading control) in 5-
day overloaded (OL) and contralateral normally loaded (NL) plantaris
muscles of Control, L-NAME, and TRIM-treated rats. B) Quantification of
phosphor(Thr389)-p70s6K to total p70s6K ratio. Values are means (SEM)
expressed relative to Control/NL mean. a = Significantly different from
corresponding NL p<0.05. b = Significantly different from Control-OL,
p<0.05.









+ control NL OL NL OL

iNOS 00*


Figure 4-5. Immunoblot assessment of iNOS protein expression in 5-day overloaded
(OL) and contralateral normally loaded (NL) plantaris muscles of Control rats.
iNOS expression was not detected in any of the samples.














CHAPTER 5
DISCUSSION

To our knowledge, this is the first study to investigate the effects of functional

overload on the up-regulation of key signaling pathways leading to increased protein

synthesis, angiogenesis, and satellite cell activation and proliferation. Our primary

observations were focused on an early time point (5d of overload) when growth factor

expression is at its peak (2). The data supports our hypothesis that NOS activity is

important for up-regulation of contractile gene expression. Specifically, skeletal a-actin

type I (slow) MHC up-regulation in the overloaded plantaris was prevented with both

non-isoform-specific inhibition of NOS activity (L-NAME) and nNOS-specific inhibition

(TRIM). Conversely, neither L-NAME nor TRIM treatment repressed the overload-

related increase in skeletal muscle mRNA expression for myogenin and the growth

factors: HGF, VEGF-120, IGF-1, and MGF. In fact, the TRIM-OL group expressed

approximately double the MGF and IGF-1 transcripts compared to Control-OL.

Consistent with increased IGF-1 signaling, NOS inhibition induced greater relative

phosphorylation of p70s6K in OL muscle. Although it appears that nitric oxide signaling

is not necessary for the up-regulation of the growth factors we measured, the data suggest

that nitric oxide may play a role in the transcriptional regulation of slow MHC, skeletal

a-actin, the phosphorylation status of p70s6k, and, perhaps, feedback control of IGF-

1/MGF mRNA during skeletal muscle overload.

Employing in vivo models to investigate nitric oxide signaling, although

physiologically meaningful, is not without consequence. Systemic non-isoform-specific









NOS inhibition (L-NAME) has physiological consequences throughout the body, and can

lead to significant effects on hemodynamics (14), muscle contractility (20), and gene

expression in non-muscle tissue (35). The majority of these systemic L-NAME effects

are thought to be secondary to eNOS inhibition and the resulting effects on blood flow

and blood pressure. In fact, the nNOS-specific isoform, TRIM, has been administered to

rats in vivo with no reported systemic side-effects (39). Since nNOS is the most abundant

NOS isoform in skeletal muscle (70), and is reportedly sensitive to muscle loading (71),

we hypothesized that this isoform accounts for overload-induced nitric oxide signaling in

the plantaris muscle. To test this possibility, and partially control for the systemic effects

of L-NAME, we treated one group of rats with daily IP injections of TRIM. Since TRIM

inhibits iNOS as well as nNOS, we confirmed by immunoblots that iNOS protein was not

expressed in NL or OL plantaris muscles (data not shown). Our data support a role for

the nNOS isoform in early adaptations to skeletal muscle overload.

Skeletal a-Actin mRNA Expression

Skeletal a-actin protein is an important component of the contractile apparatus, and

is known to be transcriptionally up-regulated during skeletal muscle hypertrophy(9).

Since adult skeletal muscle sarcomeric actin is derived from the single a-skeletal actin

gene, rather than from multiple isoforms, the regulation of this gene serves as an index of

overall contractile protein synthesis. Carson et al( 1) have reported that transcriptional

activity of the actin promoter is increased in skeletal muscle during in vivo stretch

overload. This effect is mediated by serum response factor (SRF) binding to actin

promoter (10). A recent paper (36) found that nitric oxide donors were sufficient to

induce SRF binding to a myosin heavy chain promoter element and increase promoter









activity in cultured smooth muscle cells. Our data suggest that nNOS activity is important

for induction of skeletal a-actin transcription during chronic overload.

VEGF Expression

The reduction in total VEGF mRNA level in the 5d-overloaded muscle was

unexpected, given the known capillary angiogenesis occurring in overloaded rat muscle

(54). However, the few studies reporting VEGF mRNA expression in hypertrophying

skeletal muscle show mixed results. Degens et al. (17) found no significant changes in

VEGF mRNA in hypertrophying quail muscle. Similarly, 4 wks of strength training in

human subjects, even under hypoxic conditions, did not change skeletal muscle VEGF

mRNA expression (23). On the other hand, overload of the rat plantaris for 2 wks (i.e.

2.8X longer than our 5d treatment) did increase VEGF mRNA by -50% (18).

To further characterize the VEGF mRNA response, we examined expression of the

four splice variant isoforms found in rat skeletal muscle (16) using semi-quantitative RT-

PCR and published primer sequences (16). To our knowledge, ours is the first study to

measure expression of specific VEGF mRNA splice variants in overloaded,

hypertrophying skeletal muscle. Unlike aerobic exercise, which primarily induces the

VEGF-164/5 isoform (29, 37), we found that the VEGF-120 isoform was induced in the

5d-overloaded plantaris muscle. This effect, however, was not influenced by L-NAME or

TRIM treatments.

IGF-1 Expression and Phosphorylation of p70s6 Kinase.

These results are consistent with previous findings that endogenous IGF-1 mediates

adult skeletal muscle hypertrophy, as our data shows a dramatic increase in the amount of

transcript with OL. Additionally, we show that the MGF splice variant is particularly

responsive to overload. These data support a role for nitric oxide that is either









independent of the IGF-1 axis, or downstream of IGF-1 transcription. The exaggerated

expression of IGF-1 and MGF mRNA in the overloaded plantaris of the nNOS-inhibited

TRIM group suggest the possibility of an nNOS-dependent negative feedback mechanism

controlling the load-induced IGF-1 response. IGF-1/Akt signaling in the rat kidney is

known to activate eNOS by phosphorylation and increase nitric oxide production (75).

The phosphorylation status of the NOS isoforms in overloaded skeletal muscle is

unknown, but it seems possible that IGF-1-dependent nNOS activation could produce a

feedback signal to control IGF-1/MGF expression during muscle growth.

Activation of the key translational regulator, p70s6K, is correlated to increased

protein synthesis in skeletal muscle, induced by phosphoinositide 3-kinase (PI3K) or

mechanical stretch (33). Since NOS inhibition reduces protein accumulation in

overloaded rat plantaris, we postulated that phosphorylation of p70s6K would be inhibited

in the L-NAME and TRIM groups. On the contrary, we found that OL induced

expression of total p70s6K protein in all groups, and that this corresponded to an increase

in phosphorylated p70s6K in the OL muscles. However, the ratio of phosphor/total p70s6K

indicating the relative degree of activation of the pathway was elevated only in the OL

muscles of the L-NAME and TRIM groups. This suggests that protein translation may

have been elevated in partial compensation to reduced transcriptional activity. Further

experiments are needed to directly measure effects of nitric oxide on skeletal muscle

protein synthesis rates during hypertrophy.

Future Directions

Although many of our results are unremarkable, there remain many unanswered

questions regarding the mechanisms underlying skeletal muscle hypertrophy. Therefore,

there are other pathways yet to be investigated that may play an important role in









contributing to muscle growth. We will look at two possible contributors that deserve

future attention and the possibility of interaction with nitric oxide signaling: calpain-

mediated proteolysis and calcineurin-NFAT pathway.

Calpain-Mediated Proteolysis. The calpains are a ubiquitous family of calcium-

dependent cytosolic cysteine proteases. Calpain proteolysis activity contributes to overall

protein degradation. Specifically, the calpains target proteins that are important in

linking cytoskeletal proteins together to the cell membrane (25). Nitric oxide has been

implicated as a possible regulator of calpain activity. The nitric oxide donor, sodium

nitroprusside, reversibly inactivates calpain activity via S-nitrosylation (47). Further,

NOS activity has been shown to inhibit calpain protease activity in skeletal muscle(38).

Therefore, it is also possible that protein accumulation during overload in the NOS-

inhibited groups could be hindered by an increase in protein degradation. Preliminary

data comparing protein content of cleaved ac(II)-spectrin between the groups at 5 days of

overload showed inconsistent results (data not shown). However, this could be due to a

variety of factors, including length of time between tissue harvest and data collection in

this instance. Nonetheless, calpain activity inhibition may represent an important

mechanism by which nitric oxide production facilitates hypertrophy.

Calcineurin-NFAT pathway and the slow MHC phenotype. Calcineurin is a

calcium-dependent protein phosphatase located in skeletal muscle cytoplasm that

preferentially responds to intracellular calcium concentration. Once activated, the

cascade of events leads directly to muscle growth and fiber type differentiation (65, 73).

A target for calcineurin is a member of the nuclear family of activated T-cells (NFAT),

which remains in the sarcoplasm in a phosphorylated state. Upon dephosphorylation, the









protein translocates to the nucleus and promotes transcription of genes which are

involved in hypertrophy and fiber morphology shift (40, 50). Calcineurin has been

implicated in both stimulation of type I gene expression and facilitation of type II to type

I fiber type transition. Naya and colleagues (49) concluded that activated calcineurin

induces type I gene expression via NFAT transcription. The c-Jun N-terminal kinase

(JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway has

been implicated in the rephosphorylation and subsequent resequestering of NFAT to the

cytoplasm. Interestingly, in cardiac muscle there exists a cross talk between JNK and

calcineurin-NFAT signaling such that JNK activation acts to modulate calcineurin-NFAT

signaling and inhibit cardiac growth(42). Further, nitric oxide increases NFAT nuclear

accumulation indirectly via cGMP-dependent kinase (PKG) in smooth muscle (27).

PKG, in turn, directly inhibits JNK activity, allowing NFAT to accumulate in the

nucleus. Taken together, these data suggest additional pathways through which nitric

oxide may be regulating the hypertrophy response in skeletal muscle and should be

investigated further.

Conclusions

Up-regulation of type I (slow) MHC and skeletal a-actin mRNA (presumably via

transcription) during chronic skeletal muscle overload is dependent upon nNOS activity.

Conversely, induction of growth factors and activation of protein translation (p70s6k

phosphorylation) are not dependent upon NOS activity. Nevertheless, nitric oxide

production may provide feedback control of IGF-1 and MGF signaling in hypertrophying

muscle.
















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53


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BIOGRAPHICAL SKETCH

Jeff Sellman was born in Kettering, Ohio, and grew up in Littleton, Colorado. He

graduated summa cum laude from Heritage High School in 1991. After 2 years of

undergraduate work at the University of Colorado at Boulder, Jeff took time off to pursue

other interests. This led him to the Florida Army National Guard where he was a land

combat missile system repair technician. During his time in the army, Sergeant Sellman

was an expert M16 marksman, was designated an army physical fitness leader, and

received his combat lifesaver certificate. He was awarded The Army Reserve

Component Achievement Medal and The Army Achievement Medal. Jeff returned to

school at the University of Florida, Gainesville, Florida, and graduated cum laude with a

bachelor's degree in exercise and sports sciences. He began a master's program in

applied physiology and kinesiology also at the University of Florida. He has worked for

four years as a research assistant in the Molecular Physiology Laboratory within the

Center for Exercise Science. Jeff has been accepted into the University of Florida

College of Medicine Class of 2009 and will pursue an M.D. degree.