University of Southern California Dissertations and Theses

Satellite cell proliferation: two models and two anabolic stimuli

(USC Thesis Other)

Satellite cell proliferation: two models and two anabolic stimuli

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content SATELLITE CELL PROLIFERATION: TWO MODELS AND TWO ANABOLIC STIMULI by Hans Christian Dreyer A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOKINESIOLOGY) December 2004 Copyright 2004 Hans C. Dreyer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION This dissertation would not have been possible without the love, support and sacrifice of my wife, Sarah. She is responsible for all that is good in my life. Sarah has provided me with two absolutely incredible children. They are intelligent, warm, packed with personality, and full of joy. As beautiful as Christian and Makayla are to behold, they are far more of a treasure on the inside, just like their mother. Sarah is a remarkable individual and Christian, Makayla and I are very fortunate to have her in our life. My wife is my best friend. She has sacrificed a tremendous amount for me, my career and our kids. Thank you Sarah! I love you and the kids love you. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS First, I must acknowledge the dedication and support of my mentor, Dr. Robert A. Wiswell. Dr. Wiswell has guided my development as a researcher and scientist. His many years of experience with Ph.D. students created an environment that I was able to work well in. Additionally, his career as a Department Chair, academician, and researcher has provided a base of understanding and insight into the workings of grant writing, research and departmental administration that have significantly contributed to my current and will contribute to my future success. Dr. Cesar Blanco instructed me in the powerful techniques of immunohistochemistry and enabled me to ask the “satellite cell question”, directly. Cesar has spent a good portion of the last three years with me at his side and has molded me into a skeletal muscle physiologist. During the time spent in his lab as a research assistant, I obtained a great appreciation for his depth and breadth of knowledge, some of which was passed on to me. Cesar and I have engaged in ‘lively’ discussions of skeletal muscle issues, resulting in mutual respect and understanding. Dr. Fred Sattler enabled me to ask my question in the human model. Fred generously provided me with his time and experience. He performed the history, physical, stress tests, and biopsies on all of my human subjects, requiring many hours. Moreover, Dr. Sattler’s experience and dedication to research and to his graduate students enabled me to navigate potential hurdles and roadblocks presented iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by the IRB. The human component of this dissertation would not have been possible without his assistance! I must also acknowledge the support and guidance of Dr.’s Steve Hawkins and Todd Schroeder. Not enough can be said about these two individuals. Steve made things tough but simple, asking the always important “what is your question” question. Dr. Schroeder made sure I kept my research goals realistic and feasible (i.e., achievable). Additionally, Todd and I have engaged in numerous discussions on the topics of skeletal muscle, aging, exercise (resistance and endurance), metabolism, materialism, family, and SCIENCE. I learned from Todd on the former issues and he learned from me on the latter issues. Drs Schroeder and Hawkins mentorship, guidance and support have truly been instrumental in my success. Thank you gentlemen! I must also acknowledge the generous financial support extended to me by Dr. Jim Gordon and the Department of Biokinesiology & Physical Therapy. Additionally, I must acknowledge the financial assistance provided to me by the Foundation for Physical Therapy Additionally; Promotion of Doctoral Studies II (PODS II), and the General Clinical Research Center (GCRC) grant, 5 M01 RR- 00043 from the National Center for Research Resources, NIH. I must thank Dr. Kornelia Kulig, and fellow graduate students Shruti Arya and Stephanie Babbidge who contributed significantly to the writing of manuscripts, and the dissertation. Lastly, I must thank the volunteer-subjects, without whom I would have no human component to my dissertation study. To all of you, I thank you! iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DEDICATION.................................................................................................................. ii ACKNOWLEDGEMENTS............................................................................................iii LIST OF TABLES..........................................................................................................vii LIST OF FIGURES........................................................................................................viii ABSTRACT......................................................................................................................ix CHAPTER I OVERVIEW...............................................................................................1 SPECIFIC AIMS........................................................................................................... 4 Glossary of Abbreviations............................................................................................ 5 CHAPTER II REVIEW OF THE LITERATURE.........................................................7 Satellite Cell.................................................................................................................. 7 Introduction............................................................................................................... 7 Source of New Myonuclei....................................................................................... 9 Satellite Cell Characteristics.................................................................................. 11 Satellite Cell Activation..........................................................................................13 Satellite Cell Migration.......................................................................................... 15 Are Satellite Cells Required?................................................................................. 15 Other Potential Sources of Myoblasts................................................................... 16 Satellite Cells Respond to Muscle Extract............................................................ 17 Acute Bout of Exercise and the Satellite Cell Response......................................21 Satellite Cells and Hypertrophic Stimuli...............................................................22 Satellite Cells Respond to Testosterone................................................................24 Skeletal Muscle and Sarcopenia.................................................................................29 Peak Muscle Strength..................................................................................................30 Strength versus Mass Gains in Older Adults............................................................31 Muscle Disruption.......................................................................................................33 Introduction............................................................................................................. 33 Eccentric Contractions and Muscle Disruption.....................................................34 Myogenesis and Muscle Disruption.......................................................................35 Muscle disruption and mechano growth factor (MGF)........................................36 CHAPTER III TEMPORAL EFFECTS OF TESTOSTERONE ON SATELLITE CELL PROLIFERATION IN THE MALE RAT DIAPHRAGM 37 Introduction..................................................................................................................37 Materials and Methods................................................................................................38 General Surgical Procedure....................................................................................38 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunohistochemistry........................................................................................... 40 Histological analysis.............................................................................................. 41 Statistical Analysis................................................................................................. 42 Results..........................................................................................................................42 Diaphragm Muscle Fiber Number and Fiber Cross-Sectional A rea...................42 Myonuclei............................................................................................................... 43 Satellite Cell Proliferation...................................................................................... 44 Discussion....................................................................................................................47 CHAPTER IV SATELLITE CELL PROLIFERATION IN YOUNG AND OLDER MEN 24 HOURS AFTER A SINGLE BOUT OF MAXIMAL ECCENTRIC EXERCISE............................................................................................. 51 Introduction................................................................................................................. 51 Materials and Methods................................................................................................53 Study Design............................................................................................................53 Maximal-Eccentric Exercise...................................................................................54 Skeletal Muscle Biopsy Procedure and Specimen Processing............................ 55 Immunohistochemistry........................................................................................... 56 Histological Analysis.............................................................................................. 57 Statistical Analysis.................................................................................................. 58 Results..........................................................................................................................59 Characteristics of the Study Groups...................................................................... 59 Design Characteristics............................................................................................ 60 Maximal-Eccentric Resistance Exercise................................................................61 Delayed Onset Muscle Soreness............................................................................62 Number of Muscle Fiber Analyzed........................................................................64 Fiber Type Proportions.......................................................................................... 65 Skeletal Muscle Fiber Cross-Sectional Area.........................................................66 Myonuclei/Muscle Fiber.........................................................................................67 Central Myonuclei/Muscle Fiber........................................................................... 68 Satellite Cells...........................................................................................................68 Satellite Cells Proportions...................................................................................... 73 Discussion....................................................................................................................74 CHAPTER V SUMMARY AND CONCLUSIONS.................................................. 80 Summary of Study I ....................................................................................................80 Summary of Study II...................................................................................................82 Future Directions.........................................................................................................86 REFERENCES................................................................................................................ 88 v i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 3-1 Morphological Data by Group.....................................................................43 Table 4-1 Subjects Characteristics & Blood Chemistries............................................60 Table 4-2 Muscle Tissue Data by Group and Time....................................................65 Table 4-3 Fiber Type Morphologic D ata.....................................................................66 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 2-1 Potential Sources of Myoblasts in Skeletal M uscle..................................17 Figure 2-2 Factors Effecting Satellite Cell Activity....................................................20 Figure 3-1 Myonuclear Domain....................................................................................44 Figure 3-2 Digital microphotographs............................................................................46 Figure 3-3 Satellite Cell Proportion............................................................................. 47 Figure 4-1 Averaged Peak Torque (Nm) by Group..................................................... 62 Figure 4-2 Modified Borg Scale for Delayed Onset Muscle Soreness by Group 63 Figure 4-3 Representative Digital Microphotographs................................................. 69 Figure 4-4 Digital Microphotographs of Cell Division............................................... 70 Figure 4-5 Individual Values for Satellite Cells/Muscle Fiber...................................72 Figure 4-6 Percent Increase in Satellite Cells/Muscle Fiber from Baseline............. 73 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Skeletal muscle is a dynamic tissue. It can respond rapidly to abrupt changes in use and hormone levels. The plasticity of muscle is attributed to some extent to a muscle precursor cell called the satellite cell. Satellite cells provide myoblasts necessary for repair and hypertrophy of skeletal muscle. In response to anabolic stimuli, increased loading or injury satellite cells divide and become myoblasts, ultimately forming new myonuclei. Diminished satellite cell contribution of myoblasts has been proposed as a potential source of the age-related blunting of the regenerative process and the reduced hypertrophic response to anabolic stimuli. Studies have demonstrated that anabolic-androgenic steroids increase satellite cell numbers in both rat and human skeletal muscles. Study one of this dissertation sought to determine the time-course of satellite cell activation in the rat model utilizing the diaphragm muscle to control for activation history. Serum testosterone levels were elevated to approximately 6.5x physiologic levels for zero (sham- control), seven, 14, and 28 days in gonadally intact adult male rats. We showed significant increases in average number of satellite cell number per diaphragm muscle fiber after seven and 14-days of testosterone propionate treatment. These data suggest a temporal response of satellite cell proliferation to chronic testosterone propionate treatment. Additionally, increases in satellite cells were observed without concomitant increases in myonuclear number or fiber cross-sectional area, suggesting that testosterone may increase satellite cell proliferation without activating their myogenic potential. ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The second study of this dissertation tested the hypothesis that older human muscle would respond with less satellite cell activation 24 hours after a single bout of maximal-eccentric exercise compared to young muscle. We obtained muscle biopsies from ten young (23 -3 5 years) and nine older (60 - 75 years) men, 12.5 days apart. The post-exercise biopsy was performed 24 hours after a single bout of maximal-eccentric exercise and the pre-exercise biopsy served as control. Twenty four hours after exercise, we observed a significant increase in the number of satellite cells/muscle fiber in both age groups. However, the magnitude of change was significantly greater in younger muscle. These data demonstrate greater satellite cell proliferation from young muscle. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I OVERVIEW Adult mammalian skeletal muscle represents a stable organ with little turnover of myonuclei (49, 150). Microtrauma induced by day-to-day locomotion elicit minimal renewal of the constituent multinucleated myofibers (36). Skeletal muscle satellite cells initially described by Mauro (115) in 1961, are currently thought to be the primary source of new myonuclei in postnatal skeletal muscle (36, 120,123). A small portion of satellite cells may be activated at any one time in order to replenish the estimated 1-2% of myonuclei that are replaced each week (150). Satellite cells are normally quiescent, but may be activated (i.e., go from Go to Gi in the cell cycle) in response to the appropriate anabolic (endocrine signal and/or hypertrophic loading) stimuli and become muscle precursor cells (MPC) (78). Subsequent to continued expression of factors favoring myonuclear expansion, the MPC’s will continue down the myogenic lineage, ultimately becoming myoblasts and incorporated into regenerative muscle tissue or fusing to each other to form a myotube (36, 78). Diminished satellite cell contribution of myoblasts has been proposed as a potential source of the age-related attenuation of the regenerative process (42) and muscle mass accretion associated with anabolic stimuli (107). Current anabolic therapies designed to dampen or reverse the age-related loss in muscle protein mass include, but are not restricted to testosterone treatment and progressive resistance training (PRT). Supplementation with anabolic androgenic 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. steroids (AAS) in humans has established that increases in muscle cross-sectional area (CSA) and strength are achieved with this mode of stimuli (14,15, 17, 18, 56, 121, 153-155, 176). Similarly to AAS, progressive resistance training has the desired effect of increasing muscle mass and strength in older adults, including nonagenarians (59). Some investigators have demonstrated no age-related differences after PRT in young versus older subjects (139). While other studies have demonstrated a blunted anabolic response to PRT in older muscle (73, 183). The mechanism(s) responsible for the potential differential response between age groups are not known. It is likely that attenuated responses in older persons are multi factorial resulting from genetic, lifestyle, hormonal and environmental factors that may be interdependent and act on many different levels throughout the lifespan. Studies utilizing animal models have demonstrated similar age-related attenuation in muscle mass accretion in response to anabolic stimuli (28, 34, 108). The mechanisms responsible for this apparent age-related discrepancy may be due to the reduced myogenic response demonstrated in older rat (172) and human (75) muscle after a single bout of resistive exercise. The following two studies approach satellite cell activation on two fronts: the first study address the temporal issue of satellite cell activation in response to T treatment in the male rat diaphragm. This study was conducted in response to a recent investigation by Sinha-Hakim et al., (163), who reported that in man testosterone supplementation resulted in significant dose-dependent satellite cell expansion. However, it is unknown what effects testosterone has on parameters of 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. satellite cell activation, proliferation and differentiation in systems exogenously supplemented with supraphysiologic levels of this hormone. Thus, we designed a study to test the hypothesis that satellite cell proliferation would mirror the duration of testosterone treatment over a 28-day time period. The second study was designed to test the hypothesis that satellite cells from older men would demonstrate a blunted satellite cell proliferative response to maximal-eccentric exercise relative to young men. This study would be the first conducted on humans to assess the contribution of satellite cell proliferation to the myogenic response 24 hours after the activating stimuli. Only two studies have measured the potential age related effects of resistance type exercise on satellite cell proliferation in humans (79, 142). Moreover, the results from those studies were based on biopsies obtained at the beginning and end of the treatment program, which may fail to capture age-related changes in satellite cell activation occurring early on. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SPECIFIC AIMS 1 To determine the effects of zero (Sham-Control), seven, 14, and 28 days of supraphysiologic testosterone propionate on satellite cell proliferation in the gonadally-intact male rat diaphragm. (Chapter 3) 2 To determine the amount of satellite cell proliferation in young and older muscle 24 hours after a single bout of unilateral maximal-eccentric exercise. (Chapter 4) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Glossary of Abbreviations • 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) • 5-bromo-2’-deoxyruidine (BrdU) • activities of daily living (ADL) • anabolic androgenic steroids (AAS) • androgen receptor (AR) • basic helix-loop-helix (bHLH) • bone marrow (BM) • choline acetyltransferase (ChAT) • cluster of differentiation (CD) • cross-sectional area (CSA) • delayed onset muscle soreness (DOMS) • deoxytyrosine-triphosphate (dTTP) • epidermal growth factor, (EGF) • extensor digitorum longus (EDL) • extracellular matrix (ECM) • fibroblastic growth factor, (FGF) • growth hormone (GH) • hepatocyte growth factor/scatter factor (HGF/SF) • insulin-like growth factor I, (IGF I) • levator ani (LA) • muscle precursor cells (MPC) • muscle-derived stem cells (MDSC) • myogenic-regulatory factors (MRF) • myosin heavy chain (MyHC) • neural cell adhesion molecule (NCAM) • normal donkey serum (NDS) • one-way analysis of variance (ANOVA) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. • optimum cutting temperature (OCT) • platelet-derived growth factor, (PDGF) • progressive resistance training (PRT) • room temperature (RT) • side population satellite cells (SPSC) • statistical package for social sciences (SPSS) • stretch overload (SO) • testosterone propionate (TP) • tibialis anterior (TA) • transforming growth factor (31, (TGF- p 1) • Trenbolone acetate (TBOH) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II REVIEW OF THE LITERATURE Satellite Cell Introduction The plasticity of muscle is attributed, to some extent, to a muscle precursor cell called the satellite cell. Upon muscle injury, mechanisms are initiated to restore the muscle fiber into a functional contractile apparatus once again. Satellites cells provide myoblasts necessary for repair and hypertrophy of skeletal muscle. In response to anabolic stimuli, increased loading or injury, satellite cells divide and become myoblasts ultimately forming new myonuclei. Satellite cells, named by Mauro in (115) for their location on the periphery of muscle fibers, reside within the basal lamina surrounding individual muscle fibers on the plasmalemma surface our bound by the basal lamina of the extracellular matrix (ECM) externally and by the plasma membrane of the skeletal muscle internally. Satellite cells resemble myonuclei in both appearance and size but have a poorly developed cytoplasm (71). The nucleus of a satellite cell very nearly constitutes the whole cell, leaving little room for intracellular organelles. Longitudinally, satellite cells are 8.3 pm in length in normal human tissue (182). Proportionally they constitute 30% of sublaminar nuclei in mice at birth which falls to <5% in a 2-month old adult (23). 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Satellite cells lie dormant (quiescent) until they are needed. Quiescent satellite cells reside on the periphery of muscle cells and are separated from the muscle cytoplasm by a thin cell membrane called the plasmalemma. The plasmalemma envelops the entire inner surface of skeletal muscle cells. Superficial to the plasmalemma is the basal lamina, which embryologically is laid down after the satellite cells are securely attached to the outer surface of the myotube (a muscle fiber prior to innervation by a motor nerve). This effectively sandwiches the satellite cell between the basal lamina and the plasmalemma (9). The overall consensus is that satellite cell numbers appear to decrease as a function of the age of the animal (72). Recent findings in humans have challenged the notion of an age-related decline observed in animal models. Similar to the reports from mice (42) which demonstrate no significant change with age in satellite cell proportions, Hikida et al., (79) reported satellite cell proportions of 2.4% for young (22.5 ± 5.8 years) and 2.3% for old (65.0 ± 6.0 years), Roth et al., (141), reported 2.8% for young (25 ± 3 years) and 1.7% for older (69 ± 3 years) men, Crameri et al., (45), 2.59% for young (25 ± 3 years) men, and Charifi et al., (37) who reported satellite cell proportions of 2.4% in older men (73 ± 3 years) at baseline. However, Kadi et al., (89) reported significantly higher proportions of satellite cells from young men (25 ± 3 years) relative to older men (69 ± 3) (7.1% versus 4.4%, respectively). The apparent discrepancies between studies may be due to the muscles sampled. Our data (Chapter 4) as well as those reporting similar values mentioned above (37, 45, 79, 142) were derived from biopsies of the vastus lateralis 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. muscle, whereas Kadi et al., (89) sampled tissue from the tibialis anterior (TA) muscle. Satellite cells have the capacity to self-replicate (43, 78, 166). Additionally, they have the ability to proliferate and differentiate into myoblasts (22, 82, 165, 167- 169). Myoblasts can then fuse to uninjured portions of muscle fibers, and contribute to the repair process (78, 82, 120). The essential role of satellite cells is to contribute new myonuclei to the generation (development) and regeneration (repair) of muscle fibers (71). This is important because if a muscle fiber or fibers were to be injured those nuclei within the damaged tissue will die, which removes critical genetic information required for the production of proteins critical for repair (159). Moreover, the additional DNA may potentiate the skeletal muscles ability to synthesis proteins, potentially allowing for and sustaining an increase in muscle mass (1, 3, 78). Adult mammalian skeletal muscle is a stable tissue with little nuclei turnover (150). Skeletal muscle cells are multinucleated. These myonuclei are located near the outer portion of the muscle cell, and do not have the capacity to self-replicate (they are terminally differentiated; i.e., committed to being myonuclei) (150). This means that new nuclei must come from some other source and that source is the satellite cell. Source of New Myonuclei Until recently the satellite cells were thought to be the primary source for myoblasts in skeletal muscle. Recent evidence, however, have called that notion into 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. question. Progenitor cells from bone marrow (BM) (57), the adult musculature (11), the aorta (48), and ECM (187) can under certain circumstances differentiate into myoblasts. However, the data suggest that the contribution of these non-muscle stem cells in normal skeletal muscle repair and adaptation is minor if any (36, 42). To set the stage for satellite cell activation it is important to verify that, indeed the satellite cells are the source of additional myoblasts and subsequently myonuclei. Snow et al., (166) used H-thymidine injections as a radioactive label that will be incorporated into mitotic cells during replication (DNA copies) to differentiate the source of new myonuclei. H-thymidine is a radioactive label that will become incorporated into the DNA strand during replication. First, he injected pregnant rats at 16, 18, and 20 days gestation followed by three injections into the neonatal rats postpartum. These rats were allowed to mature (4-5 weeks) and then killed (166). Examination of various skeletal muscles revealed labeled myonuclei; however, satellite cells were not labeled (166). These findings suggest that the labeled myonuclei were recently synthesized. In Addition, Dr. Snow injected tracer in 18- day-old rats followed by sacrifice 10 hours later (166). Muscles from these rats revealed labeled satellite cells but no labeled myonuclei, indicating that satellite cells periodically self replicate and therefore take up the label. We know that post-mitotic myonuclei do not self replicate and are therefore unable to take up the labeled marker. These results suggest that labeled myonuclei were the result of fusion of myoblasts during embryonic development such that once differentiation was complete what was left was labeled myonuclei (166). However, when 18-day-old 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rats were injected with tracer 5-bromo-2’-deoxyruidine (BrdU) only satellite cells were labeled. Indicating that originally, the neonatal rats’ satellite cells were labeled, and with subsequent fusion of myoblasts to form muscle fibers and subsequent differentiation, these myoblasts now transitioned into myonuclei. Satellite cells from these rats were not labeled either because they lost their label during mitotic division, which dilutes the label or because those satellite cells that were labeled differentiated into myoblasts, which are now myonuclei. Additionally, Snow et al., (166) injected tracer into rats one-hour prior to mincing of sections of various rat skeletal muscles in order to determine which cells would subsequently take up the tracer. Eight to 16 hours after mincing, labeled mononucleated cells, located between the basal lamina and degenerating plasmalemma were observed (166). These mononucleated cells were presumed to be presumptive myoblasts, and because only satellite cells were labeled with the tracer, it seems probable that these presumptive myoblasts originated from these satellite cells (166). Satellite Cell Characteristics Satellite cells express c-Met (tyrosine kinase receptor) and M-cadherin protein during quiescence but do not express markers of committed myogenic cells (myoblasts) such as the myogenic-regulatory factors (MRF) Myf-5 or MyoD (44). Myogenic regulatory factors are a group of basic helix-loop-helix (bHLH) transcription factors (stimulate the possibility of protein production) that are expressed by satellite cells upon activation. Basic helix-loop-helix transcription 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. factors have a central loop structure that serves as a DNA-binding/dimerization basic region and two ends which are both activation domains with a-helix orientation (106). MyoD is able to stimulate transcription only when two or more homodimers of MyoD are bound to multiple E boxes; which are DNA binding sites with a 5’- CANNTG-3’ consensus sequence that are found in the regulatory region many muscle-specific genes (106, 113). Activated satellite cells express either MyoD or Myf-5 initially and soon thereafter co-express both MRFs, which cause determination of the satellite cell to become a myoblast (147). Continued activation by secondary MRFs’ myogenin and MRF-4 (expressed by the satellite cell) cause terminal differentiation and fusion of the myoblast to existing muscle fibers (147). In the embryonic model, these myoblasts would fuse together to form a myotube, which is a muscle fiber precursor. Once a myotube is innervated, it becomes a muscle fiber. Post embryonic myoblasts will fuse with pre-existing muscle fibers and contribute (growth/hypertrophy) its DNA to the existing muscle fiber genetic pool or replace those myonuclei that were lost due to muscle disruption. Replacing the number of myonuclei brings the muscle fiber back to homeostasis. However, increasing the number of myonuclei allows for more genetic material, and subsequent potential for protein production to maintain a larger muscle volume (3, 38,159). This is an integral part of our discussion because it has been suggested that myonuclei can govern only a limited volume of cytoplasm, and if that volume expands so too must the number of myonuclei. This is the ‘myonuclear domain 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. theory’ of skeletal muscle adaptation (74), which was originally proposed by Cheek (38) as the ‘deoxyribonucleic unit’. This is a critical point because if this theory were incorrect, additional myonuclei would not be necessary, as surviving myonuclei would simply increase production of the necessary proteins to make up for the loss (38). However, as will be demonstrated in a later paragraph, satellite cell number, as well as absolute myonuclear number, increases with hypertrophy, thus maintaining a relatively stable cytoplasm to myonuclear ratio. The next step is to determine what type of myofiber will be expressed by these satellite cells. In other words, will a satellite cell produce fibers with the same characteristics of the previous fiber? Experiments with extracted satellite cells originally from either a fast fiber (type II) or a slow fiber (type I) have demonstrated that irrespective of origin (i.e., fiber type) expression is embryonic myosin heavy chain (MyHC) > neonatal MyHC > type I MyHC (137). Neither fast MyHC isoform (Ha or Ilb/x) were expressed. These are satellite cells that are cultured, without the influence of nerve-growth factors such as neuregulin, which other researchers (54, 137) have shown to be essential for fast MyHC expression. This brings up an important concept. Muscle-nerve interaction is a continuous interdependent relationship. Researchers must address the interdependence of muscle and nerves in order to gain better understanding of what is transpiring in vivo. Satellite Cell Activation Bischoff, (22) determined that the mitogenic activity of minced muscle was due to a high-molecular-weight polypeptide. Tissue extract was passed the through a 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30,000 Dalton cut-off filter and applied to the same techniques as described above for tissue extract. Additionally, Bischoff tested the possibility of transferrin as the possible mitogenic activator due to his prior work (21, 22) which demonstrated transferrin as essential for chick embryo myogenic activity. He was able to conclude that transferrin had no effect on satellite cell activity. Since this seminal study by Bischoff, (22) a number of researchers have investigated various factors (either muscle derived or other) to elucidate the identity of satellite cell activator(s) (22). Of the examined growth factors (fibroblastic growth factor, (FGF); insulin-like growth factor I, (IGF I); platelet-derived growth factor, (PDGF); transforming growth factor p i, (TGF-pi); epidermal growth factor, (EGF) only hepatocyte growth factor/scatter factor (HGF/SF) stimulated quiescent satellite cells to transition from G0 to Gi phase of the cell cycle in living muscle and culture (175) and also to stimulate their proliferation in injured muscle (118). Hepatocyte growth factor is a located in the ECM of undamaged, intact skeletal muscle however, post-injury it is found on the satellite cell surface where it binds to the c-Met receptor (175). To test whether HGF was indeed responsible for satellite cell activation, anti-HGF antibodies were used to block their ability to bind with the c-Met receptor. BrdU labeled myonuclei decreased from pre-anti-HGF antibody administrations levels of -60% to control levels (175). This work demonstrated that by blocking HGF activation capacity, satellite cells were unable to contribute new myonuclei and thus BrdU labeling levels remained at control levels despite the presence of muscle disruption. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Satellite Cell Migration As mentioned earlier activated satellite cells have been shown to migrate to areas of muscle damage both within the muscle fiber housing the satellite cell as well as to adjacent injured muscle fibers (20). The exact mechanism remains unknown; however, TGF-p has been demonstrated to exert powerful chemoatractive control over satellite cell movement. Chemotaxis is the ability of factors to cause certain cells (such as white blood cells and macrophages during injury) to migrate to the zone of injury. Remarkably, along with attracting the satellite cell, TGF-P also exerts a negative control on satellite cell proliferation (20). Proliferation capacity is reduced by ~50-60% in the presence of TGF-P (6). Bischoff, (20) demonstrated that TGF-P prevents cells from transitioning from the Go to Gi phase (satellite cell activation), which he postulated was due to the fact that motility is suppressed in mitotic cells. This sets up a situation where the attractor (i.e., TGF-P) will summon satellite cells (chemotaxis) to the focal point of injury, while at the same time preventing them from proliferating until they reach the zone of injury. Are Satellite Cells Required? Irradiation experiments have indicated that satellite cells are essential for hypertrophy to take place. Rosenblatt et al., (136, 138) and Phalen et ah, (129) demonstrated that low-level radiation, which destroys the satellite cells replicative potential but leaves the rest of the muscle intact, resulted in a muscle that is unable to respond to appropriate stimuli with a hypertrophic response. Radiation will prevent satellite cell mitosis by causing breaks in the DNA, which will prevent DNA 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. replication. Myonuclei, however, are post-mitotic (non-replicating) and the damage done by irradiation has a greater chance of occurring in non-coding sections of DNA rather than the protein-coding sections of DNA, constituting roughly 1 -2% of the total DNA. Thus the chances of radiation damaging skeletal muscle protein coding sections are very small; whereas any damage done to DNA will most likely prevent replication that requires intact DNA strands (106). Both groups used synergistic ablation to cause functional overload of the extensor digitorum longus (EDL), in growing mouse (135), adult rat (138), and rat soleus (129) muscle. Groups were divided into those receiving irradiation and overload and those receiving overload alone. Compensatory hypertrophy was achieved in muscles not irradiated, while those that were irradiated did not hypertrophy. In fact there was a tendency for the cross-sectional area to decrease as compared to controls in the irradiated muscles. Other Potential Sources of Myoblasts Myoblasts within skeletal muscle may arise from multiple sources under experimental conditions. Progenitor cells from BM (57), the adult musculature (11), the aorta (48), and ECM (187) can under certain circumstances differentiate into myoblasts. Within skeletal muscle there are three potential sources of myoblasts currently muscle-derived stem cells (MDSC), side population satellite cells (SPSC) and satellite cells themselves described above (120, 128). Muscle-derived stem cells are thought to possess several differentiative possibilities and may be a skeletal muscle satellite cell predecessor (see Figure 2-1, from (55)) (33, 81). Muscle SPSC cells can potentially, if given the correct environmental cues, become hematopoietic 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells, skeletal muscle, or satellite cells after transplantation into irradiated muscle tissue. Moreover, at least one of these potential sources resides within the interstitial space of skeletal muscle and is positive for the cluster of differentiation (CD) cell surface marker CD34 (170, 171). Lastly, the majority of data suggest that the contribution of these non-muscle stem cells in normal skeletal muscle repair and adaptation is minor if any (24, 36, 42, 128). Bone Osteogenic Marrow Osteocalcin C alcium p ho sp h ates A lkaline phosphatase activity | ■Hematopoietic n H P Adipogettic I j j i i * CD45 E f i t I ^ R S j Saluratcd ncutml lipids J W WyrioM E l g k . ™ j SKH PPARy Lymphoid C/EPBot (SPcells) v Skeletal jV ivQ gcniC ' Chondrogenic Myosin isofgm s JH L , Sutfonated ulycosatnino«l>C)ins M ill's Collagen type 1 1 (Satellite eelB)otherMyogenic Lineages Smooth muscle ?; Cardific muscle ? Figure 2-1 Potential Sources of Myoblasts in Skeletal Muscle (Fano et al., 2004) Satellite Cells Respond to Muscle Extract It has been demonstrated that breaks in the sarcolemma are required for satellite cell activation and proliferation (22). This sets up a situation where in order 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for satellite cells to contribute to muscle cell repair two things must happen. First, some type of damage must occur to the sarcolemma. This may come from strength training, running, walking, etc., where a micro-tear of the muscle cell membrane occurs. Second, the satellite must receive some signal, which stimulates it to initiate a proliferative cascade that results in new myonuclei as well as at least one new satellite cell to replace the activated one. Satellite cells have been demonstrated to activate while incubated with crushed muscle tissue (22). Initially, satellite cells were cultured in basal medium for a period of four days, during this time no change in satellite cell number was seen. Next, separate cultures had various tissues added to determine what type of tissue was necessary for satellite cell activation. Intact muscle added to the culture had no significant affect on satellite cells (22). This was also the case when liver, lung, heart and kidney tissue, as well as when no tissue was added to the culture for an additional four days (22). However, when crushed skeletal muscle was added a significant increase was seen in the number of labeled cells. Interestingly, satellite cell activity increased, although not significantly, with the addition of minced heart and kidney extract (22). This may be due to variance in cultured cell activity or it may be that these two tissues contain quantities of satellite cell activator(s) proportional to change in satellite cell proliferation. Bischoff, (21) was able to demonstrate that satellite cells appear to be under negative influence of myofiber plasmalemma (21). He was able to show this by subjecting the satellite cell to various cultures in which satellite cells were in contact 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with combinations of myofiber, basal lamina, and substrata all in the presence of muscle extract. As was the case for the cell cultures above, the addition and removal of the plasmalemma resulted in an attenuated proliferation of satellite cell number and significantly increased proliferation of satellite cells, respectively. Bischoff, (21) suggest that the ECM exerts negative proliferative control on the satellite cells by contact inhibition which is removed when the myofiber is absent or killed by Marcaine in the presence of minced muscle extract (21). Thus, removal of the plasmalemma alone is not sufficient to induce proliferation of satellite cells (19), the addition of mytogen-activator (minced muscle extract), which exerts positive control, is essential for proliferation of satellite cells. Bischoff, (21) suggests this as an explanation for why nearby satellite cells fail to respond to focal injury, while satellite cells within the zone of focal injury respond by proliferation and differentiation into myoblasts for muscle tissue repair (19). Contact with the plasmalemma appears only to exert a negative influence, since Bischoff, (21) was able to induce satellite cells in contact with the plasmalemma to proliferate in the presence of high concentrations of muscle extract. This would suggest that negative contact control of the basal lamina may be overcome in situations where a greater degree of muscle injury has occurred some distance away from the satellite cell. Thus, local satellite cell proliferation is mediated by local muscle damage and differentiation, whereas, if the degree of muscle damage is substantial (ever increasing exposure of satellite cells to damaged muscle) then distant satellite cells may be recruited to provide tissue repair (157). Indeed mechanisms exist that cause 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. satellite cells to migrate within a myofiber (between the basal lamina and muscle fiber) to an injured zone as well as between myofibers. This suggests a dose response of satellite cells to varying degrees of muscle disruption. It remains that several levels of control exist in the regulation of repair of muscle damage by satellite cells and many different factors can exert influence on satellite cell activation (Figure 2-2). Immune Respons Other Factors LIF iL -e O ftfaer !S testosterone nitrfcoxldte macrophages: neutrophils IGF-11 FGF HGF neurotransmittors neurotrophic factors T EGF IGF-1 IGF-II FGF HGF TGF-p Vasculature Autocrine Factors Figure 2-2 Factors Effecting Satellite Cell Activity (Hawke and Gary, 2003) 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acute Bout of Exercise and the Satellite Cell Response Recent work looking at the effect of one vs. multiple bouts of a muscle damaging activity (decline treadmill running) has revealed that there is an apparent dose response. Smith et al., (164) studied satellite cells proliferation after control, one, two, four or seven days of consecutive decline treadmill running in rat muscles. New myonuclei (originating from satellite cells) were determined by constant infusion into the circulation of these rats with BrdU. Newly synthesized DNA thymine is partially replaced by BrdU, which is in high concentration added to the plasma medium (164). This label is converted to the corresponding triphosphate and will be incorporated into DNA in place of thymine which compete with deoxytyrosine-triphosphate (dTTP), bromouracil base pairs like a thymine and will be inserted into the new DNA strand opposite an adenine in the template strand (new DNA strand). Results indicated a significantly greater proliferation after two days of a single bout versus one day only and four and seven consecutive days of decline treadmill running (164). After two consecutive days of this activity there where 3.4 ± 0.7 labeled per 1,000 myonuclei, however, after one day of activity there where 2.8 ±3.2 labeled per 1,000 myonuclei (164). These results indicate that continued muscle damaging activity at two versus one day results in a greater proliferative response and that continued daily activity for four and up to seven consecutive day’s results in no further increase in satellite cell proliferation. The data from that study suggests that satellite cell proliferation returned to control levels despite continued muscle 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. damaging activity at four and seven days (164). This may represent a saturation limit whereby beyond a certain amount of activity, in this case two consecutive days of decline treadmill running, results in no further satellite cell proliferation. Additionally, it may simply reflect an acute phase adaptation on the part of the rat such that repeated bouts of the same activity resulted in greater synchronization of motor unit firing patterns (i.e., motor learning response to unaccustomed exercise). Histological evidence of muscle damage was significantly greater in the rats seven days after one bout decline treadmill running (7.4 ± 1.7%) vs. controls (4.2 ± 1.7%) or following seven days of daily decline treadmill running for (4.7 ± 0.8%). Similar rational as above applies to these results; either the proliferating satellite cells (daily group) were able to attenuate the number of damaged fibers due to greater amounts of repair, or that continued daily decline treadmill running failed to illicit muscle damage (i.e., motor learning response). It remains that these results suggest a physiologic rationale for promoting the continuation of a muscle-damage activity following the initial exercise. Similar results have also been found by others (94, 181). Satellite Cells and Hypertrophic Stimuli In a study directly addressing satellite cells and aging in an animal model Carson et al., (34) determined that after two weeks of stretch overload, aged quail muscle responded with equal satellite cell activation as the young quail. This study provides evidence that aging does not attenuate the activation of satellite cells per se; 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. however, this model utilized the method of stretch overload to induce muscle damage (34). Although, the model of stretch overload has been used extensively to produce muscle disruption in animal models, the mechanism of action differs from that of eccentrically induced muscle damage in that the muscles are not activated in the stretch overload model. Additionally, studies utilizing the stretch-overload model have not addressed the specificity of damage in terms of fiber type susceptibility, which has been reported on by Lieber et al., (101) to be three times more likely in the Type lib fibers. Lowe et al., (108) designed a study to determine the effects of age on MRF upregulation induced by hypertrophic stretch overload (SO) for six, 24, or 72 hours in adult, middle-aged, aged, and senescent quails. Myogenic regulatory factors MyoD and MRF4 but not myogenin expression were reduced in the petagalis muscles of older quails (aged and senescent) after SO, which they proposed as a possible source of the differential response of older muscles fail to respond to hypertrophic stretch overloading stimuli with increases in cross-sectional area (CSA), which are observed in the muscles of younger animals (108). The data from Lowe and colleagues (108) are in conflict with Carson & Alway (34) who suggest that aging does not attenuate the satellite cell response, which may be reflected in differences in timing. Lowe (108) studied muscles after six, 24 and 72 hours of SO whereas Carson studied two weeks of SO, both in aged and adult quail petagalis muscle. It may be that initial events, up to 72 hours, in the aged quail 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. population fail to respond significantly to SO and require the additional 11 days to produce significance. Or it may be that by 14 days adult quail petagalis satellite cell numbers return to a quiescent phase and thus masks any real changes on the part of the aged quail at this time point. Kadi and Thomell (93) have demonstrated similar findings in human application of strength training in female trapezius muscle. Over a ten-week training period nine subjects provided biopsy samples that were analyzed for myonuclear and satellite cell numbers as well as percent increase in fiber CSA. Myonuclear number increased 70%, satellite cell number increased 46% and CSA increased 36%. Mean myonuclear number per fiber cross-section was correlated with CSA (r = 0.6) (93). These researchers also demonstrated a correlation between myonuclear number and satellite cell number (r = 0.5) (93). This supports results by Gibson and Schultz (69) who found satellite cell distribution to vary by fiber type (Type I fibers had significantly greater satellite cells than Type II fibers). The reason for this discrepancy has been theorized by Roy et al., 1999 to be due to the greater enzymatic synthesis required of oxidative fibers as well as the greater protein turnover rates in slow versus fast fibers. On average Type I fibers have smaller nuclear domains followed by Type Ha and Ilb/x, respectively (i.e., myonuclei number follow this pattern; Type I>IIa>IIb) (145). Satellite Cells Respond to Testosterone The ability of testosterone to stimulate skeletal muscle mass accretion is a common observation (154, 155). Testosterone treatment in young hypogonadal 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. males increases lean tissue mass (16) and muscle strength (16), which involves augmenting myofibrillar protein synthesis rates, demonstrated in older men (56). Ultimately if muscle mass is positively influenced, these changes may be preceded by myonuclear accretion, involving the increase in myonuclear numbers to maintain a greater muscle cytoplasmic volume (i.e., the myonuclear domain hypothesis (74)). As mentioned above additional nuclei arise from satellite cell differentiation into myoblasts, which will fuse to each other forming a myotube or to the existing muscle fiber becoming centrally located nuclei (36). Because both final components of the neuromuscular system, the a- motomeuron and skeletal muscle fibers both express the androgen receptor (AR), the effects of anabolic-androgenic steroids (AAS) may potentially modulate function via either structure (27). Blanco et al., (26) have shown that testosterone treatment in the supraphysiologic range can increase choline acetyltransferase (ChAT) mRNA levels in motorneurons of the adult male rat spinal cord. If indeed the increased ChAT mRNA expression leads to protein product, this could potentiate ChAT activity levels at the axon terminal and increase presynaptic acetylcholine synthesis (27). Tucek et al., (180) have shown that ChAT activity is unaffected in the soleus muscle (primarily composed of Type I myofibers) by supraphysiologic testosterone levels suggesting that AAS induced modulation of may be fiber type dependent. Moreover, Blanco et al., (27) has demonstrated that Type IIx and lib fibers decrease their susceptibility to neuromuscular transmission failure after treatment with AAS. These data potentially suggest that Type II myofibers are more amenable to AAS 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. levels. A recent report by Monks et al., (119) demonstrated that AR positive myonuclei in the levator ani (LA) muscle in close proximity to the neuromuscular junction are positively influenced by testosterone treatment, potentially providing support for a Fiber Type specific effect of AAS observed by Blanco et al., (27). Furthermore, increased specific twitch and tetanic tension have been demonstrated by AAS supplementation in the rat diaphragm (27) which was observed without concomitant increase in fiber CSA. The above reports suggest that AAS cause modification in the neural component of the neuromuscular system in the rat diaphragm muscle. Potential behavioral modification due to AAS treatment must be addressed when using testosterone as an anabolic agent in research studies. Testosterone propionate (TP) is a Class I AAS and has been demonstrated in animal models to effect behavior in the form of increased aggression (39). Thus, a study designed to assess the effects of AAS on the temporal response of satellite cells must utilize a muscle that will not be affected by the AAS induced behavioral modification, such as the diaphragm, which will not be stimulated more or less by the phrenic nerve. Previous studies have reported that AAS, such as testosterone can increase satellite cell numbers in male (124-126) and female (85-87, 178, 179) rat LA and in the human vastus lateralis muscle (163). Studies conducted by Nnodim et al., (124- 126) have demonstrated the permissive role of testosterone in satellite cell expansion post denervation in the male rat LA muscle. However, the LA muscle is unique in that it has a greater number of AR relative to other skeletal muscle (12). 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Additionally, the LA muscle requires testosterone to be present during denervation induced satellite cell expansion, whereas the EDL does not require the presence of testosterone for satellite cell expansion also due to denervation (126). In a study conducted by Joubert and Tobin (85) a time-dependent response was reported for satellite cells as a result of cyclohexane propionate testosterone (0.25 mg/100 g of body weight) administration in the female rat LA muscle. That study elevated testosterone levels by injecting a slow-release long-duration (5-week) acting AAS which stimulated satellite cell proliferation, becoming significant within 48 hours and subsequently reaching peak numbers by day-3 that were -three times control values (85). Satellite cell numbers remained stable until day six after which a significant decrease was observed between days six and nine and again between days 15 and 30 whereby satellite cell numbers had returned to control values (85). In that study, myonuclear numbers significantly increase twenty-four hours after the satellite cell expansion (e.g., on day-3), suggesting a portion of the satellite cells had progressed down the myogenic pathway and became incorporated into the muscle tissue as nuclei. Significant increases in satellite cell and myonuclei number have been reported to occur in men after 20 weeks of weekly injections of supraphysiologic testosterone enanthate (163). Similar increases in satellite cell and myonuclear number have been observed in the rat LA muscle; a highly sexually dimorphic and androgen sensitive muscle, of rats (86, 126). Indeed, testosterone has been demonstrated to be necessary for the activation of satellite cells subsequent to 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. denervation (126). These results demonstrate that satellite cells may be activated by testosterone and suggest that at least part of the anabolic effects of testosterone (i.e., increase skeletal muscle mass) are mediated by satellite cells. However, it may be that the observed increase in satellite cell numbers was a result of cumulative responses from each individual injection or potentially the result of a proliferative response to the last injection (163). Indeed, evidence for such was presented by Joubert and Tobin (85) mentioned above. Cell culture studies have uncovered information of the direct actions of testosterone on satellite cells. Direct addition of Trenbolone acetate (TBOH), a testosterone analog (17(3-acetoxy-3oxoestra-4,9,l 1-triene) to porcine satellite cells for 72 hours does not result in increased proliferation and has a negative effect on differentiation (177). Additionally, increased labeling of myonuclei was observed in three of four muscles studied without concomitant increase in muscle CSA. It was only after examining the major semimembranosus muscle that those investigators observed both an increase in muscle mass and myonuclei accretion (177). These changes were observed despite the fact that AR has been shown to be upregulated in all of the nuclei of clonally derived satellite cells and fibroblasts (51). Interestingly, satellite cells from rats treated with TBOH were more responsive in cell culture to FGF and IGF-I than satellite cells from untreated rats (177). 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Skeletal Muscle and Sarcopenia Skeletal muscle represents the largest protein reservoir in the human body. It makes up approximately 45% (55) of total body weight and contains between 50 - 70% of all proteins in the human body (132). About 19% of skeletal muscle is protein (186). Muscle protein serves as an endogenous source of amino acids that can be mobilized for the use in other parts of the body during times of malnutrition, trauma, surgery, and illness (132,144). The mobilization of amino acids involves the tightly regulated process of catabolism. The balance between anabolism and catabolism determines the protein mass and hence the muscle mass status of the individual. Sarcopenia is the involuntary decline in lean muscle mass and strength that occurs with aging (144). The length of human life currently averages between 75 to 78 years and may increase to 85 years within the next two decades (98). Adding years to the lifespan, however, raises the question of whether or not these years will be healthy years or years lived consisting of compromised physical, mental, and social function (52, 98). The projected increase in the number of older adults will, no doubt, increase the number of persons suffering the burden of sarcopenia. It is not necessarily the result of disease but is often observed in aging adults. Sarcopenia increases the risk of disability and loss of functional capacity in the elderly. As sarcopenia progresses, activities of daily living and mobility are further impaired, which may result in osteoporosis, falls, fractures, thrombophlebitis, pulmonary embolism, isolation, depression, and other adverse consequences. It is estimated that 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14% of persons between the age of 65 to 75 require assistance with activities of daily living (ADL) and that percentage increases to 45% in persons over 85 years of age (98). The issue of sarcopenia that relates to this proposal is one of involuntary muscle loss. At the whole body level, muscle loss results in the decrement in force producing capability. At the cellular level muscle loss leads to a decline in the available muscle protein stores. The status of muscle protein is influenced by a variety of factors including genetic regulation, nutrition, physical activity, hormones (growth hormone (GH), IGF-I, cortisol, androgens, insulin) and the health status of the individual. Each of these factors independently or in combination may play a role in sarcopenia. Understanding the mechanisms involved with muscle mass accretion will enable us to formulate methods to stop or reverse the process of age- related muscle mass loss. Peak Muscle Strength Strength will be defined herein as the result of both central and peripheral factors contributing to the maximal force generating capacity of skeletal muscle. Muscle mass and strength generally peak between the ages of twenty and thirty-five years of age (117). Approximately 30% of voluntary strength is lost between 50 and 70 years of age (100). After the age of sixty sharp declines in strength are accompanied by declines in muscle mass (104,109). In the Copenhagen Heart Study, knee extensor strength in healthy 80-year-olds was about 30% less than in 70- 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. year-olds (47). Strength is closely associated with muscle mass (114). Reports indicate that muscle strength is greatly diminished by the 6th and 7th decade of life and that a more rapid drop in strength occurs thereafter. It has been suggested that, besides illness and undernutrition, the lack of muscle activity (i.e., a sedentary lifestyle) contributes significantly to loss of muscle strength and exerts a major influence on sarcopenia (77, 185). Strength versus Mass Gains in Older Adults Progressive resistance training in older adults has been shown to increase strength and to some extent muscle mass, in older adults, including nonagenarians (59). Fiatarone et al., (59) demonstrated improvements in strength in nine institutionalized frail nonagenarians who underwent eight weeks of high-intensity strength training that was as great as 174% with only a 9% increase in mid-thigh muscle CSA. They reported a remarkable capacity for change in a population that may not be included by many in a potential pool of strength trainable subjects. The ability for older subjects to increase muscle strength is of significance because the number one predictor of functional status in this age group is muscle strength (58). However, as mentioned above, adequate stores of amino acids are also necessary for the immune response during acute illness, as well as the recovery phase of surgery (132, 143). Thus, maintaining mass is beneficial in terms of preventing possible accidents that lead to hospitalization as well as recovering from the immobilization that may result if it were to occur. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Although older individuals can demonstrate significant gains in strength and mass, the magnitudes of the increases in muscle mass (hypertrophy) is significantly less than those observed in younger adults despite equal relative training intensities and strength gains (73, 183). Hakkinen et al., (73) demonstrated that older men (mean age 66 years) were able to increase maximal isometric leg strength by 36% compared to young (mean age 42 years) who also increased strength by 36% after six months of heavy-resistance training. While quadriceps CSA significantly increased by 5% in the young men; older men demonstrated a 2% (not significant) increase. Similar results were found by Welle et al., (183). His group assessed strength and hypertrophic gains by MRI in nine young (age range 22-31, five male and four female) and eight old (age range 62-72, four male and four female) after three months of progressive resistance training. They found pre-training muscle quality to be less in the older group for all muscle groups tested (16% for elbow flexors; 40% for knee flexors; and 19% for knee extensors). Increases in CSA after training were significantly less in the older group for elbow flexors (22% in young, 9% in old) and knee flexors (8% in young, 1% in old), but no differences were observed for knee extensors (4% in young, 6% in old) (183). Studies utilizing animal models demonstrate similar age related attenuation in muscle mass, as well as strength accretion (28, 34, 108). Blough et al., (28) examined the effects of functional overload for eight weeks in adult and old rats. Baseline peak isometric tetanic tension was 83% less in the old compared to the 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. young. At the end of 8 weeks the overloaded muscle of the young animal increased in wet mass by 53% and type I, Ha, and Ilx/b fiber CSA increased by 91, 76, and 103%, respectively. No increase in wet mass or whole muscle CSA were observed in the muscles of the old rats (28). Muscle Disruption Introduction The mechanism by which eccentric loading initiates satellite cell activation remains unknown. Current theories suggest that sarcolemma damage and intramyofibrilar protein disruption are primary factors initiating the repair process (60). As has been review above, HGF and factors released by the ECM of muscle can stimulate the c-Met tyrosine kinase receptor located on satellite cells, initiating activation. Overt cellular disruption visualized by light (102) and electron microscopic techniques have demonstrated that the magnitude of muscle damage is greater after eccentric loading versus either isometric or concentric in animals. However, recent studies identifying muscle damage at the light (45) and electron microscope (140, 142) have failed to identify a link between the degree, if any, of muscle damage and the magnitude of satellite cell response in humans. Characteristics of muscle disruption at the cytoskeletal level include I band broadening, Z-line streaming or smearing, or even total obliteration. In addition A- bands within the affected sarcomeres demonstrate lateral shift to one side or the other. Z-disc streaming appears to be a primary consequence of muscle damaging 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. exercise. The initial injury from stretching or muscle use appears to be the result of mechanical damage to the cytoskeleton of individual sarcomeres. This results in the typical disarray of previously well organized A- and I-bands. Additionally, Lieber et al., (103) have demonstrated reductions in desmin staining post eccentrically induced muscle damage. Desmin is a Z-disc protein, which aids in maintaining the structural integrity of the Z-disc. Lieber et al., (103) have demonstrated muscle damage by the absence of desmin staining as early as 15 minutes post eccentric muscle action. Previous research has demonstrated that eccentrically based resistive exercise results in skeletal muscle damage (131). Muscle damage as a result of eccentric contractions in humans occurs on the descending limb of the sarcomere length- tension relationship curve, a point at which tension development in titin proteins are increasing exponentially (95, 96). Eccentric Contractions and Muscle Disruption. It is widely accepted that muscle damage is associated with exercise involving eccentric type contractions. Most people are familiar with the post exercise soreness with transient muscle disruption/inflammation from eccentric contractions, commonly refereed to as delayed onset muscle soreness (DOMS), which peaks 24-48 hours after strenuous or unaccustomed exercise (60). It has been well accepted that physical activity involving eccentric muscle actions result in more severe muscle disruption histopathologicaly (10, 29), enzymatic (97), and indirectly via symptoms (60). 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Progressive resistance training or strength training has been proven to increase muscle strength and cross-sectional area (116). The increase in muscle strength and size is largely due to an increase in the cross-sectional area of muscle (i.e., increase in contractile protein) (8). Strength training involves lifting and lowering of weights. Lifting weight involves shortening of muscle fibers while producing force (e.g., concentric contraction), while lowering the weight involves lengthening of muscle fibers while producing force (e.g., eccentric contraction). Resistance training involving eccentric contractions consistently result in greater muscle strength and hypertrophic gains than concentric or isometric (muscle produces force without changing length) alone, in human (40, 41, 53) and animal (7, 35,108) models. Additionally, eccentric contractions, versus concentric or isometric contractions, are associated with greater amounts of muscle disruption (61-63, 67) and inflammation (31, 32). Thus, greater muscle disruption may explain the greater gains in muscle strength and mass associated with eccentric type contractions. Indeed, initial muscle disruption has been correlated to gains in strength (r = 0.857) (161). A common observation of muscle fiber disruption is Z-disc streaming and smearing as observed with light (62) and electron microscopy (46). Myogenesis and Muscle Disruption. Myogenesis or muscle regeneration after disruption has been well established. Satellite cells, which normally lay dormant between the basal lamina and sarcolemma of muscle cells, become activated with muscle disruption. This is accomplished when factors like HGF is released from the basal lamina, where they 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reside until disruption, and bind with the c-Met receptor of satellite cells (160, 173- 175). Activated satellite cells proliferate and differentiate into myoblasts (muscle nuclei precursor cells) (84, 135, 138, 158). This allows for an increase in the number of muscle nuclei in order to produce proteins necessary for repair and hypertrophy, the latter in order to maintain myonuclear domain characteristics (5, 74, 90, 145, 146, 162, 164). The mechanisms involved in myogenesis have yet to be defined however, a clear association between muscle disruption and inflammation has been demonstrated (60). Muscle disruption and mechano growth factor (MGF) Recently, Hameed et al., (75) demonstrated significant increases in MGF mRNA levels in young, but not in elderly subjects 2.5 hours after high-resistance exercise. Hameed’s work stemmed from the observation of Owino et al., (127) who demonstrated that when rat soleus and plantaris muscle were overloaded through synergistic tendon ablation, there appeared to be a reduction in the ability of older muscle to upregulate MGF mRNA. Baseline (no intervention) expression levels of IGF-I at various ages have not shown a differences in either mRNA or peptide level expression. Furthermore, a age related inverse relationship between synergistic hypertrophy of 60%, 35%, and 20% in the young, mature, and old rats, respectively has previously been demonstrated (127). Insulin-like growth factor I receptor mRNA levels were found to be significantly increased in the overloaded muscle of the young, however, no such increase was observed in the mature and old rats (127). 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III TEMPORAL EFFECTS OF TESTOSTERONE ON SATELLITE CELL PROLIFERATION IN THE MALE RAT DIAPHRAGM Introduction Satellite cell proliferation and differentiation provide myonuclei for repair and hypertrophy of skeletal muscle (36, 78) and provide the majority of muscle precursor cells (36, 42) in the adult. The addition of new myonuclei by satellite cells to existing muscle fibers associated with anabolic stimuli (e.g., resistance training, testosterone supplementation), is necessary for sustained myofibril synthesis and increases in fiber-CSA resulting in an increase in force generating capacity. Current therapies designed to enhance skeletal muscle mass such as AAS have clearly established that increases in muscle CSA and strength are achieved with this mode of stimuli (14, 15, 17, 18, 56, 121, 176). A recent investigation by Sinha-Hakim et al., (163), demonstrated in sedentary men, that a twenty week course of weekly testosterone injections significantly increased satellite cell numbers in the vastus lateralis muscle in a dose dependent manner. It may be that the observed increase in satellite cell numbers was a result of cumulative responses from each individual injection or potentially the result of a proliferative response to the last injection. Evidence for the latter scenario may be provided by a study conducted in the female rat by Joubert and Tobin (85). They performed a detailed analysis of satellite cell proliferation subsequent to a single injection of cyclohexane propionate 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. testosterone (0.25 mg/100 g of body weight) (85). In that study satellite cell numbers significantly increased within 24 hours (85). Proliferation continued reaching peak values by day-3 and was maintained until day-6, demonstrating significant declines by day-9, which was the next time point assessed, ultimately returning to baseline between days 15 and 30. That study provided the first (and to our knowledge only) evidence for a temporal response of satellite cells to a single injection of testosterone (85). However, due to the highly sexually dimorphic and androgen-sensitive nature of the LA muscle in addition to the female phenotype, the ability to generalize these data requires further testing in a male model. The purpose of this study was to determine the temporal effects of continuous supraphysiologic levels of testosterone propionate (TP) on satellite cell proliferation in the diaphragm muscle of sedentary gonadally intact male rats. The diaphragm muscle was chosen to reduce the potentially confounding effect of testosterone- induced modulation of locomotor activity. Materials and Methods General Surgical Procedure All protocols were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee at the University of Southern California. Surgical techniques were done under aseptic conditions with post-surgical recovery carefully monitored. Adult male Sprague- 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dawley rats (body weight: 250-275 g) were used throughout the study. Food and water were provided ad libitum and a 12/12 light/dark cycle was maintained utilizing automated switching devices. Pathogen-free animals were randomly assigned to two groups, sham-operated controls or those treated with TP. Continuous release capsules were made from Silastic™ tubing (inner diameter: 1.57 mm; outer diameter: 3.17 mm), checked for leakage and pre equilibrated in sterile saline for 24 hours prior to implantation. Sham-controls (n = 5) were subcutaneously implanted with a single empty 45-mm capsule. Male rats receiving testosterone treatment were subcutaneously implanted with two 45-mm capsules filled with TP (TP; 4-androsten-17(3 -ol-3-one 17 propionate; Steraloids) under ketamine:xylazine anesthesia (ketamine: 50 mg/kg; xylazine: 10 mg/kg). Similar methods have been used previously by us (27) and others (126). Blanco et al., (27) have shown that serum testosterone levels are elevated approximately 6.5 times greater than physiologic conditions after 28 days of testosterone treatment (27). Controls (n = 5) and TP-treated rats [treatment duration: 7 (n = 5), 14 (n = 5), or 28 (n = 5) days] were deeply anesthetized with sodium pentobarbital (150 mg/kg) and killed by transcardial perfusion with saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline. At this time, previously implanted capsules were removed and inspected to ensure residual TP remained. The diaphragm was removed, cryoprotected in 20% sucrose solution in PBS until processed for immunohistochemical analysis. 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunohistochemistry Previously cryoprotected diaphragm muscle was frozen in isopentane cooled to the temperature of crushed dry-ice and mounted in optimum cutting temperature (OCT). Diaphragm muscle samples were sectioned transversely and sections were cut at a thickness of 10 pm in a cryostat (Hacker Instruments, England) maintained at -25°C (APPEDIX A). Alternating serial sections were processed for the immunohistochemical identification of satellite cells (M-cadherin), and myonuclei (4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI)), and muscle fiber boundaries (i.e., Laminin). Two diaphragm tissue sections from each condition (i.e., Control, seven, 14, and 28 days of TP treatment) were mounted in that order on a Superfrost Plus slide (Fisherbrand). This was performed in duplicate. Each section was washed in 0.050 M Tris-buffered saline (TBS; pH 7.40), followed by incubation in TBS containing 0.3% Triton X-100 with 4% normal donkey serum (NDS) for one hour in a humidified chamber at room temperature (RT) to block non-specific binding sites. Sections were subsequently placed in a primary antibody medium containing polyclonal goat anti-M-cadherin antibody (directed against the amino terminus of human M-cadherin (identical to the corresponding mouse sequence; Santa Cruz Biotechnology; final dilution: 1:500), and polyclonal rabbit anti-laminin antibody (DakoCytomation; final dilution 1:2000), in TBS containing 0.3% Triton X-100 with 2% NDS for overnight at RT. One diaphragm tissue section from each animal was incubated in a primary antibody medium lacking the anti-M-cadherin and 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. anti-laminin antibodies to determine the degree of non-specific binding of the secondary antibodies. The diaphragm tissue sections were subsequently washed in TBS, followed by a two hour incubation in 0.03% Triton X-100 and 2% NDS containing Cy3- conjugated donkey anti-goat IgG (to visualize M-cadherin) and Cy2-conjugated donkey anti-rabbit IgG (to visualize laminin; both at a final dilution of 1:330; Jackson Immuno Research), at RT. The tissues were then washed and incubated in a solution containing DAPI (300nM) in TBS for five minutes at RT, followed by a final wash in distilled deionized water and cover slipped while wet with Gel/Mount™ mounting medium (BI0MEDA corp., Foster City, CA). Histological analysis The diaphragm tissue sections from each animal were visualized using a Nikon Eclipse E800 microscope equipped for epifluorescence microscopy (Nikon, Japan) connected to a Spot-2 digital camera (Diagnostics Instruments). A set of three images of the same visual field were digitized to visualize M-cadherin, or MyHC- slow (Cy3-fluorescence), DAPI (UV excitation), and laminin (Cy2-fluorescence). A total of 10 different visual fields from each diaphragm were digitized. Images were analyzed using a computer-assisted image analysis system (MetaMorph Imaging Software, Universal Imaging). Muscle fiber boundaries were determined using the laminin immunofluorescence. Nuclei lying within the laminin outline were considered to be myonuclei unless there was M-cadherin immunofluorescence associated with it. Cells positive for the M-cadherin 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. immunofluorescence were counted as satellite cells. M-cadherin positive staining often demonstrated a crescent shape and was localized to the section of the satellite cell membrane apposed to the sarcolemma, which is similar to published reports (50, 105, 133,148, 184). Myosin heavy chain slow isoform staining was used to determine fiber type. All satellite cells were found to be located internal to the laminin defined muscle fiber boundary. All of the analysis was performed under blinded conditions. Statistical Analysis Data were analyzed using the Statistical Package for Social Sciences (SPSS) program, version 11.0 for Windows. Data were analyzed by one-way analysis of variance (ANOVA) to determine group differences for each variable of interest. When a significant F-value was noted a Bonferroni post hoc analysis was performed to locate differences between groups. Data are reported as mean ± SD. Significance was predetermined at the p < 0.05 level. Results Diaphragm Muscle Fiber Number and Fiber Cross-Sectional Area No significant differences were observed for the number of diaphragm muscle fibers analyzed between groups (F = 0.38, p = 0.77) (Table 3-1). Table 3-1 also depicts the fiber CSA of the diaphragm muscle fibers by group. No significant changes in fiber CSA were observed (F = 0.43, p = 0.74). Additionally, a subset of muscle fibers (Sham-control = 221, 7 days = 289, 14 days = 237, and 28 days = 247 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. total muscle fibers analyzed per group) reacting positively to the myosin heavy chain-slow (MyHC-slow) antibody were analyzed for F-CSA; no changes were observed between groups (Sham-control = 586.3 ± 96.5, 7 days = 592.8 ± 104.9, 14 days = 620.9 ± 120.3, and 28 days 617.2 ± 75.2) (F = 0.15,/? = 0.93). Table 3-1 Morphological Data by Group Treatment Group Number of Muscle Fibers Fiber CSA (pm2 ) Myonuclei/ Muscle Fiber Satellite Cell/ Muscle Fiber Sham-Control (n = 5) 583 ± 164 832 ±215 0.93 ± 0.17 0.03 ±0.01 TP; 7-days (n = 5) 572 ± 77 844± 130 0.97 ±0.13 0.08 ±0.03* TP; 14-days (n = 5) 5 5 6 ± 109 819± 107 1.07 ± 0.16 0.11 ±0.03** TP; 28-days (n = 5 ) 513 ± 7 7 917 ± 121 1.03 ±0.07 0.06 ± 0.02 ANOVA- p -\alue 0.77 0.74 0.31 0.001 Data are presented as Mean ± SD. * Significantly greater than Sham-Control. ** Significantly greater than Sham-Control Group and 28 days of Testosterone Treatment Group. Myonuclei No significant differences were observed for the number of myonuclei between groups (Sham-control = 525 ± 111,7 days = 556 ± 124, 14 days = 598 ± 127, and 28 days = 556 ± 58) (F = 0.50, p = 0.69). When the number of myonuclei were expressed relative to the total number of muscle fibers (i.e., total myonuclei 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. #/total muscle fiber #), no significant differences between groups were observed (F = 1.30,p = 0.31) (Table 3-1). In addition, when the myonuclear number was expressed as ratio of fiber volume (pm3 ) to myonuclei (i.e. myonuclear domain, which is equivalent to the total area of cytoplasm “governed” by a single myonucleus (74)), no significant difference between groups were observed (F = 1.15,/) = 0.36) (Figure 3-1). a > o 3 C O >. E 11.500 10.500 9.500 8.500 7.500 6.500 5.500 4.500 3.500 2.500 1.500 500 Control (n=5) 7-Day (n=5) 14-Day (n=5) 28-Day (n=5) Days of Testosterone Treatment Figure 3-1 Myonuclear Domain Mean ± SD bars. Satellite Cell Proliferation. Visual micrographs representative of a 14-day animal are shown in Figure 3-2. One way analysis of variance revealed significant differences between groups for satellite cell numbers (Sham-control = 17 ± 6; 7 days = 47 ± 22; 14 days = 60 ± 17; and 28 days = 30 ± 8) (F = 8.07, p = 0.002). Post hoc analysis revealed significantly greater satellite cell numbers in the seven and 14 days of TP treatment groups relative to 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sham-control as well as between the 14 and 28 days TP treatment groups. A significant between group difference by ANOVA occurred for the number of satellite cells per muscle fiber (F = 9.35,p < 0.001). Post hoc analysis demonstrated significantly greater numbers of satellite cells per muscle fiber in the seven and 14 days of TP treatment groups relative to sham-controls as well as greater numbers of satellite cells per muscle fiber in the 14 days of TP treatment group versus the 28- days of TP treatment group (Table 3-1). Lastly, ANOYA revealed a significant difference in satellite cell proportions defined as; [satellite cell/(satellite cells + myonuclei)] x 100 between groups (F = 11.73,p < 0.001). Post hoc analysis determined greater satellite cell percentages in the seven and 14 days of TP treatment groups relative to controls as well as greater satellite cell percentages in the 14 days of TP treatment group relative to the 28-day TP treatment group (Figure 3-3). 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-2 Digital microphotographs. Diaphragm muscle fibers from a 14-Day TP treated male rat labeled for M-cadherin (A; Cy3- fluorescence), Nuclei (B; DAPI) and laminin (C; (Cy2-fluorescence). Arrowheads point to muscle fibers with satellite cells (M-cadherin positive). Calibration bar is equivalent to 50 pm.* Denotes muscle fiber shown at greater magnification (Figure 3-2C inset) to clearly depict M-cadherin identification of a satellite cell. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 10 a > o 3 C O ^ 5 S + o V) G O . 1 T Control (n=5) 7-Day (n=5) 14-Day (n=5) 28-Day (n=5) Days of Testosterone Treatment Figure 3-3 Satellite Cell Proportion Mean ± SD * Significantly greater than Sham-Control ** Significantly greater than Sham-Controi and 28 days of TP treatment Discussion The results of the present investigation demonstrated a temporal proliferative response of satellite cells in the diaphragm muscle of sedentary gonadally-intact male rats to chronic supraphysiologic elevation of circulating testosterone levels. Satellite cells numbers were significantly elevated for at least 14 days and had returned to baseline levels by day-28. Our findings are in agreement with and extend the results of Joubert and Tobin (85) which clearly demonstrated a proliferative response of satellite cells of the LA muscle of female rats to a single injection of testosterone that demonstrated a shorter duration time-dependent increase. The seemingly sustained increase in satellite cell numbers reported by Sinha-Hikim et al. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (163) may be a result of the cyclical changes in circulating testosterone levels. Interestingly, testosterone dependent satellite cell proliferation in the diaphragm muscle did not result in increases in fiber CSA or myonuclear density (fiber volume/myonuclei). Altogether, these observations suggest that testosterone treatment results in satellite cell proliferation independently of a significant increase in respiratory muscle activity. Rat diaphragm muscle fiber CSA was not affected by supraphysiologic testosterone treatment. This result is in agreement with previous studies on the effects of AAS on diaphragm muscle fiber CSA (25, 27, 66). Myonuclear numbers were similarly unaffected by elevated testosterone levels, partially explaining the lack of fiber CSA change and consequently the stability of the myonuclear domain throughout the study. In contrast, Joubert and Tobin (85), and Sinha-Hikim et al., (163) reported testosterone dependent increases satellite cell number (transient in the case of Joubert and Tobin; (85)) in fiber CSA and myonuclear expansion as a result of AAS treatment in the female rat LA and the male human vastus lateralis muscles, respectively. We have previously hypothesized that the lack of AAS dependent effect on diaphragm muscle fiber CSA may be due to the lack of a significant change in the respiratory neuromuscular activity (27). Whether testosterone supplementation has an overall effect on neuromuscular activity of the female LA or on lower extremity muscles in humans has not been determined. However, castration of male rats does significantly decrease the number of reflexive erections and thus results in decreased neuromuscular activity that is associated with LA 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. muscle atrophy (80). Preliminary analysis of the LA muscles from the TP treated male rats used in this study clearly demonstrates both an increase satellite cell proliferation and fiber CSA (unpublished observations). The results of the present study suggest that significant AAS dependent changes in neuromuscular activity may be necessary for AAS activation of terminal myogenic differentiation of satellite cells. An alternative explanation for our results is that TP elevation has caused a decrease in the half-life of the existing myonuclear population. This would potentially abolish any fiber CSA increases and mask any increases in absolute myonuclear numbers. In other words, the myonuclear replacement rate of 1-2% per week reported by Schmalbruch and Lewis (150) may increase as a result of AAS. Kadi and colleagues (91) analyzed biopsy samples from strength-trained athletes who abused AAS and compared them to strength-trained athletes who did not. They reported a five-fold greater incidence of centrally located nuclei in muscle cells from those athletes who abused steroids, which they suggested was due to greater fiber turnover (91). Potentially these data may provide evidence for a TP induced increase in replacement rates for the existing myonuclear pool. Clinically, the relevance of providing AAS to augment the rehabilitative potential in humans is appealing (14, 153). These data assimilated with previous reports may provide evidence for the utilization of AAS as a primer for satellite cell recruitment concomitant with rehabilitative therapy. Indeed, reports from Schroeder et al., (151, 152) suggest that the greatest increases (approaching 90%) in muscle 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. strength and mass in men supplemented with androgens are achieved within the first six of 12 weeks of treatment. It would appear that a short course of AAS may be sufficient to maximize anabolic stimuli for use in clinical application, potentially for use to hasten recovery of muscle mass lost due to inactivity or to attenuate age- associated sarcopenia. The present findings extend our current knowledge to include satellite cell proliferation in the rat diaphragm and provide support for our previous investigation (27) and studies performed by others (66), which have demonstrated no significant change in diaphragm CSA as a result of testosterone supplementation. Studies have demonstrated the requirement of skeletal muscle hypertrophy on satellite cells during anabolic stimuli (129, 136, 138). Here we show that satellite cell proliferation alone does not lead to those changes in the rat diaphragm muscle. Interestingly, AAS do not appear to affect satellite cell proliferation in vitro (177). Whether, satellite cells in vivo respond directly to AAS or are activated to proliferate by local increases in other myogenic signaling molecules remains to be determined. Further studies are required to determine the potential contribution of activation history and the influence of intermittent versus continuous AAS on parameters of satellite cells during initial treatment intervention. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV SATELLITE CELL PROLIFERATION IN YOUNG AND OLDER MEN 24 HOURS AFTER A SINGLE BOUT OF MAXIMAL ECCENTRIC EXERCISE Introduction Satellites cells provide the precursor cells necessary for repair and hypertrophy of skeletal muscle (42, 78, 129). In response to anabolic stimuli, and/or muscle damage, these cells divide and become myoblasts, ultimately forming new myonuclei (36, 78, 188). The addition of these myonuclei to the existing muscle cell allows for and maintains increases in CSA in response to anabolic signals. Diminished satellite cell contribution of myoblasts has been proposed as a potential source of the age-related blunting of the regenerative process (42) and the reduced hypertrophic response to anabolic stimuli (71, 107). Progressive resistance training has been demonstrated to increase strength and muscle mass in older adults, including nonagenarians (59). However, the magnitude of the increases in skeletal muscle mass observed in older adults may be significantly less than that observed in younger individuals (73, 183). Studies utilizing animal models have demonstrated similar age-related attenuation in muscle mass accretion in response to anabolic stimuli (28, 34, 108). The mechanisms responsible for this apparent age-discrepancy may be related to the reduced 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. myogenic response demonstrated in older rat (172) and human (75) muscle after a single bout of hypertrophic exercise stimulus. Few studies in humans have been conducted on satellite cell proliferation in response to anabolic stimuli (45, 79, 93, 112, 142, 163). Of those, only two measured the potential age related effects of resistance exercise on satellite cell proliferation (79, 142). Moreover, their results were based on biopsies obtained at the beginning and end of the resistance training program. For example, a study conducted by Hikida et al., (79) measured satellite cell proportions at the beginning and end of eight and 16 weeks of lower extremity strength training in young and older men, respectively. They reported no change in satellite cell proportions (approximating 2.4%) with the exercise regardless of age (79). However, direct comparisons between age groups from that study are complicated by the discrepancy in training durations (79). Roth et al., (142) demonstrated a significant expansion of satellite cell numbers following nine weeks of heavy resistance strength training in young and older men and women (142). Their results also demonstrated a significant age by gender interaction that was due to a greater proliferation of satellite cells in older women (142). The authors attributed that differences were the result of delayed response of satellite cell expansion from those women, possibly due to delayed activation in response to the exercise stimulus (142). Biopsies obtained after eight (79), nine (142) or 16 (79) weeks of resistance exercise have failed to demonstrate age-related differences in the proliferation of satellite cells. However, a major component of the myogenic response to anabolic 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stimuli in skeletal muscle may occur within hours to days of the initial stimulus (4, 30, 45, 110, 112, 130). Therefore, our goal was to test the hypothesis that satellite cells form both young and older muscle would increase in number but that the older muscle would respond with a less robust proliferation of satellite cells at 24 hours after a single bout of maximal-eccentric exercise. Materials and Methods Study Design. This was a prospective study to determine the effects of a single bout of maximal-eccentric exercise on satellite cell proliferation 24 hours after the exercise. Potential subjects provided informed written consent that was approved by the Institutional Review Board of the Los Angeles County-University of Southern California. Subjects had to be male either 21-35 years or at least 60 years of age and not currently resistance training. Each subject underwent a complete history and physical examination and had screening blood tests performed to determine eligibility. Subjects with cardiac abnormalities (heart failure, myocardial infarction, or angina), resting blood pressure >180/94 mmHg, active inflammatory conditions, neuropathies, untreated endocrine abnormalities (e.g., diabetes, hypothyroidism), inability to perform lower extremity exercise or receiving anticoagulation were excluded. Shortly after the study was initiated, the protocol was amended to test for serum testosterone levels. Men in the older group underwent a screening 12-lead EKG and blood pressure monitored stress test to achieve at least 85% of their 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. predicted maximal heart rate in order to minimize risk of sub clinical ischemia during eccentric exercise. All procedures involving subjects for this study were conducted at the General Clinical Research Center (GCRC) of LAC/USC County Hospital except for the maximal-eccentric loading which was performed in the Clinical Exercise Research Center, located in the Center for Health Professions Building on the Health Sciences Campus of the University of Southern California. Two biopsies were performed. The first biopsy (Pre-Exercise biopsy) served to generate baseline values for variables of interest including muscle fiber CSA, myonuclear and satellite cell numbers. The Pre-Exercise biopsy was conducted between 11-15 days prior to the second biopsy (Post-Exercise biopsy) (APPEDIX C). Both biopsies were performed on the same muscle approximately two to four cm apart. The Post-Exercise biopsy was preceded in time (24 hours) by a single bout of unilateral maximal-eccentric exercise of the dominant quadriceps muscle group. Subjects were closely monitored for signs/symptoms of adverse reaction to the biopsy or suture (APPENDIX D). Maximal-Eccentric Exercise. Single-leg maximal-eccentric isokinetic loading was performed on the dominant leg using a KinCom dynamometer (Kin-Com, Chattanooga, TN) performed at 60°/second. Each subject was familiarized with the equipment prior to the exercise. During the maximal eccentric loading a shoulder harness, hip restraint and thigh strap (exercised leg) were utilized to secure the subject to the device. A strap was also secured distally on the dominant leg at the level of the load cell. The 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. load cell was positioned three cm proximal to the talocrural joint. The intervention consisted of an initial set of 12 maximal repetitions with the subject performing the eccentric component while the investigator performed the concentric portion. This was followed by five sets of 16 repetitions, for a total of six sets; each set was separated in time by 120-180 seconds. Each subject was asked to complete a modified Borg scale (6 - 20) rating his muscle discomfort or DOMS immediately after the exercise (zero), six, 24, 48, and 72 hours after the single bout of maximal- eccentric exercise. Skeletal Muscle Biopsy Procedure and Specimen Processing. Each subject had two muscle biopsies obtained from the vastus lateralis of the dominant leg. All biopsies were conducted using a five mm Stille muscle biopsy needle (Micrins Surgical, Inc., Lake Forest, IL) using the percutaneous needle biopsy technique (13) with applied suction. The biopsy site was approximately 18 cm proximal to the patella, approximating the midline of quadriceps muscle group. The Post-Exercise biopsy was performed at a distance of three to four cm, distal or proximal (randomly assigned) from the Pre-Exercise biopsy. Approximately 100- 175 mg of muscle tissue was obtained from each subject. Muscle tissue samples were immediately rinsed with pre-chilled normal saline and dissected free from connective and adipose tissue. Muscle sections were mounted on blocks of cork (1.0x1.0x0.5 cm; length by width by height) with optimum cutting temperature compound pre-chilled by ice water. Once appropriate orientation was achieved the tissue-block was quickly frozen in isopentane cooled to 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. freezing by liquid nitrogen. Cryoprotected tissue samples were stored at -80°C until processed. Immunohistochemistry. Tissue sections were cut at 10 pm thickness in a cryostat maintained at -25°C. Tissue sections from each condition (i.e., Pre-Exercise biopsy and Post-Exercise biopsy) from the same subject were mounted in that order on a Superfrost Plus slide (Fisherbrand). Two tissue sections for Pre-Exercise biopsy and Post-Exercise biopsy were cut and oriented on each slide. Two subjects from each group were represented per slide. A minimum of eight tissue sections were arranged per slide and this was performed in triplicate. Muscle tissues were washed in 0.050 M Tris-buffered saline (TBS; pH 7.40), followed by incubation in TBS containing 0.3% Triton X-100 with 4% normal donkey serum (NDS) for one hour in a humidified chamber at room temperature (RT) to block non-specific binding sites. Sections were subsequently placed in a primary antibody medium containing monoclonal mouse anti-CD 5 6 antibody directed against the neural cell adhesion molecule (NCAM) located on satellite cells, (identical to the Leu 19 antigen (83, 99)) [Clone MOC-1, (DakoCytomation, Carpinteria, CA); final dilution: 1 TOO], and polyclonal rabbit anti-laminin antibody directed against laminin (extracellular matrix protein) (DakoCytomation; final dilution: 1:2000), and mouse anti-MyHC-slow antibody directed against muscle fibers expressing myosin heavy chain-slow isoform (Sigma, Saint Louis, MO; final dilution 1:2000), in TBS containing 0.3% Triton X-100 with 2% NDS overnight at 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RT. One slide from each experiment was incubated in a primary antibody medium lacking the anti-CD56 and anti-laminin antibodies to determine the degree of non specific binding of the secondary antibodies. Muscle tissue sections were subsequently washed in TBS, followed by a two hour incubation in 0.03% Triton X-100 and 2% NDS containing Cy3-conjugated donkey anti-mouse IgG (to visualize NCAM, or MyHC-slow) and Cy2-conjugated donkey anti-rabbit IgG (to visualize laminin; both at a final dilution of 1:330; Jackson Immuno Research), at RT. The tissues were then washed and incubated in a solution containing the nuclear marker 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes) in TBS for five minutes at RT, followed by a final wash in distilled deionized water and cover slipped while wet with Gel/Mount™ mounting medium (BI0MEDA corp., Foster City, CA) (APPEDIX F & G). Histological Analysis. Muscle tissue sections from each subject were visualized using a Nikon Eclipse E800 microscope equipped for epifluorescence microscopy (Nikon, Japan) connected to a Spot-2 digital camera (Diagnostics Instruments). A set of three images of the same visual field were digitized to visualize NCAM or MyHC-slow (Cy3-fluorescence), DAPI (UV excitation), and laminin (Cy2-fluorescence). Different visual fields of the tissue were digitized in a way to ensure that each muscle fiber was captured one time only. Images were analyzed using a computer- assisted image analysis system (MetaMorph Imaging Software, Universal Imaging). 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Muscle fiber boundaries were determined using the laminin immunofluorescence as a template. Nuclei lying within the laminin boundary (i.e., sub-laminar nuclei) were determined to be myonuclei unless there was NCAM immunofluorescence associated with it. Sub-laminar nuclei positive for the NCAM immunofluorescence were counted as satellite cells as per published reports (37, 83, 88, 91-93,111, 156). Qualification of satellite cells was as follows: first, the cell had to stain positively for NCAM; second, each NCAM positive cell must be encapsulated by the basal lamina, demonstrated by the laminin antibody; and third, each NCAM positive cell had to have a nuclei associated with it as demonstrated by DAPI. There were no incidences where a NCAM positive cell was located outside the basal lamina boarder. The NCAM antibody stains both activated and quiescent satellite cells (83, 156), and has been used previously in human tissue (37, 89, 111, 112, 134). The processing, analysis, and statistical treatment of tissue sections were performed by the same investigator. Thus, both grouping and biopsy sample were known to the investigator (i.e., the investigator was not blinded). Statistical Analysis. Data were analyzed using the Statistical Package for Social Sciences (SPSS) program, version 11.0 for Windows. Repeated-measures analysis of variance (ANOVA) was used to identify between and within group differences and to determine if an age by time interaction effect occurred. When significance was obtained, independent or paired /-tests (two-tailed) were used for between and within group differences, respectively. Baseline characteristics (blood samples and tissue 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. values) were analyzed by independent /-test. Data are reported as mean ± SD. Significance was predetermined at the p < 0.05 level. Results Characteristics of the Study Groups. Twenty-one subjects were found to be eligible and were enrolled in the study. Inadequate muscle tissue was not available from either the Pre-Exercise biopsy or Post-Exercise biopsy in one subject of each group. Thus, 19 subjects were included for the analysis. Subjects in the two groups were generally comparable except the older group had significantly lower hemoglobin, and albumin, (t = 2.2%, p = 0.036) and (7 = 3.28, p = 0.004), respectively (Table 4-1). Unexpectedly, there was no significant difference between groups for testosterone levels (t = 1.65,/? = 0.12) (Table 4-1). Physical characteristics and blood chemistries are reported in Table 4-1. 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-1 Subjects Characteristics & Blood Chemistries Measure Young Men (n = 10) Older Men (n = 9) Independent f-test p value Age (years) 28 ± 5 68 ± 6 <0.001 Weight (kg) 78 ± 13 82 ± 19 0.60 BMI 25± 3 26 ± 5 0.44 Hemoglobin (g/dL) 15.2 ±0.6 14.4 ± 0.9 0.036 BUN (mg/dL) 16.5 ±3.5 17.5 ±2.3 0.47 Albumin (g/dL) 4.4 ± 0.2 4.1 ± 0.2 0.004 AST (U/L) 27.2 ± 6.6 26.4 ± 5.8 0.80 ALT (U/L) 45.4 ± 13.2 39.8 ± 8.0 0.28 LDH (U/L) 157 ±24.9 163 ± 19.4 0.54 CK (U/L) 211 ± 9 9 148 ± 73 0.14 TSH (uIU/mL) 1.51± 0.51 2.48 ±2.52 0.29 Testosterone (ng/dL) 535± 101* 434 ± 123 0.12 Data are presented as Mean ± SD Mean testosterone values for 6 of the 10 young subjects. Abbreviations: BMI, Body Mass Index (kg/m2); BUN, Urea Nitrogen, Blood; AST, Aspartate Aminotransferase; ALT, Alanine Aminotransferase; LDH, Lactate Dehydrogenase; CK, Creatine Kinase. TSH, Thyroid Stimulating Hormone Design Characteristics. The number of days between Pre-Exercise biopsy and Post-Exercise biopsy biopsies was not significantly different between groups (12 ± 3 and 12 ± 1 days) for 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. young and older men, respectively {t = 0.19, p = 0.85). Additionally, there was no significant difference between groups for the number of hours between the maximal- eccentric exercise and the Post-Exercise biopsy (24 ± 0.5 and 24 ± 0.4 hours) for young and older men, respectively (t = 0.21, p = 0.83). Maximal-Eccentric Resistance Exercise. There was a significant between group difference in averaged peak torques (Nm) (F = 5.82,/? = 0.027) (Figure 4-3). There was no significant difference within groups (F = 1.59,/? = 0.23) or change over time (F = 0.98,/? = 0.34). Independent t- test revealed significant differences between groups for the average of sets one and two (269 ± 56 Nm and 208 ± 56 Nm) (t = 2.40,/? = 0.028) and for the average of sets three and four (268 ± 50 Nm and 209 ± 51 Nm) (t = 2.56, p = 0.021), but there was no significant difference for the average of sets five and six (250 ± 53 Nm and 206 ± 48 Nm) (t = 1.90,/? = 0.08) for young and older men, respectively (Figure 4-3). 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sets 1 & 2 Sets 3 & 4 Sets 5 & 6 ■ Young (n = 10) ■ Older (n = 9) Figure 4-1 Averaged Peak Torque (Nm) by Group. Average values for sets 1 & 2, 3 & 4, and 5 & 6 * Significantly greater than Older Group Delayed Onset Muscle Soreness Nine of the young and eight of the older subjects completed a modified Borg scale relating DOMS (APPENDIX E). There was a significant between group difference by repeated measures ANOVA (F = 4.92,/? = 0.042). Independent f-tests revealed that there was no significant difference between groups at; zero hours, 10.2 ± 4 (range = 6-17) young, and 9 ± 2 (range = 6-11) older (t = 0.84,p = 0.42); six hours, 10.8 ± 3 (range = 7-15) young, and 9.9 ± 2 (range = 6-12) older (t = 0.77,/? = 0.45); 48 hours, 9.9 ± 3 (range = 7-17) young, and 7.6 ± 1.5 (range = 6-11) older (t = 1.94, p = 0.07); and 72 hours, 6.7 ± 3 (range = 6-10) young, and 6.6 ± 1 (range = 6-10) older subjects (t - 0.94, p = 0.36). However, Independent f-test showed the DOMS score for the young group at 24 hours to be significantly greater 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. than the older group (11.2 ± 2 young, and 8.9 ± 2 older men) (t = 2.75, p = 0.015). Additionally there was a significant within group difference (F = 9.24,p - 0.008) but no interaction effect (F = 0.005,/) = 0.94). Paired samples t-test was used to determine where the within group differences existed. Young group means were significant for six versus 72 hours (t = 3.59,p = 0.007), 24 versus 72 hours (I = 4.82, p = 0.001), and 48 versus 72 hours (t = 3.82,p = 0.005). But young group means were not significantly different for zero versus 72 hours (t = 2.1 \,p = 0.068). Older group means were significant for zero versus 72 hours (t = 2.82,p = 0.026), six versus 72 hours (t = 4.33,p - 0.003), 24 versus 72 hours (t = 7.18,/? < 0.001), and 48 versus 72 hours (t = 5.29,p = 0.001) (Figure 4-2). a > O rj ® ^ 03 o % < /> ifi C (O o g T 3 < D 5. o jo to 0 Q 20 18 16 14 12 10 8 6 0 hr 6 hr 24 hr 48 hr 72 hr Hours after Maximal-Eccentric Exercise -Young (n = 9) — ♦— Older (n = 8) Figure 4-2 Modified Borg Scale for Delayed Onset Muscle Soreness by Group * S ig n ific a n tly g re a te r th a n o ld e r m en . ** Significantly less than six, 24 and 48 hours for Young group and zero, six, 24 and 48 hours for Older group. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Number of Muscle Fiber Analyzed. The number of muscle fibers analyzed satellite cell data was significantly different between the groups (F = 9.46,/? = 0.007). The number of muscle fibers analyzed was not different within groups (F = 0.05, p = 0.83) and there was no interaction effect (F = 0.22,/? = 0.64) (Table 4-2). Independent t-tests revealed a trend towards significance between groups for the number of muscle fibers analyzed at Pre-Exercise biopsy (t = 2.06, /? = 0.055) and a significant difference at Post- Exercise biopsy (t = 2.63, p = 0.018) (Table 4-2). The number of muscle fibers analyzed for fiber type comparisons was not significantly different between groups (t = 1.77,/? = 0.095) (Table 4-3). 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-2 Muscle Tissue Data by Group and Time cMean Gro up Time Point (muscle fibers analyzed) Fiber CSA (pm2 ) Myonuclei/ Muscle Fiber Central Myonuclei/ Muscle Fiber Satellite Cell/ Muscle Fiber b Satellite Cell Proportion s Change for Satellite Cell Proportio ns You ng (n = 10) Pre-Exercise biopsy (101±23) Post- Exercise biopsy (108±33) 6443 ± 994 6 2 4 0 ± 1162 2.56 ± 0 .6 2.72 ± 0 .5 0.03 ± 0.03 0.06 ± 0 .1 0 0.07 ± 0.02 0.18 ± 0.05*f 2.86 ± 0 .6 6.05 ± 1.4*t 3.2 ± 1.3 J Old er (n = 9) Pre-Exercise biopsy (134±37) Post- Exercise biopsy (132±17)§ 5134 ± 1176** 5175 ± 868*** 2.34 ± 0 .4 2.31 ± 0 .4 0.03±0.02 0.04 ± 0.03 0.07 ± 0.02 0.10 ± 0.02* 2.81 ± 0 .7 4.12 ± 1.0* 1.3 ± 0 .6 Data are presented as Mean ± SD * Significantly greater than Bxl ** Significantly less than Young at Pre-Exercise biopsy *** Significantly less than Young at Post-Exercise biopsy t Significantly greater than Older Group at Post-Exercise biopsy | Significantly greater than Older Group mean change for satellite cell % § Significantly greater number of muscle fibers analyzed than Young at Post-Exercise biopsy bSatellite Cell Proportions = [Satellite Cell/(Satellite Cell + Myonuclei)] x 100 c Mean Change = Mean (Post-Exercise biopsy - Pre-Exercise biopsy) for Satellite Cell Proportions by group Fiber Type Proportions Fiber type proportions were not significantly different within the young group (/ = 0.74, p = 0.48) but was significantly different within the older group (t = 3.00,p = 0.017) (Table 4-3). The fiber type proportions did not change between Pre- and Post-Exercise biopsy for the young (F = 1.28,p = 0.27) or older groups (F = 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.00,;? = 0.33). Myosin heavy chain-slow proportions were significantly different between groups (t = 2.46, p - 0.025) (Table 4-3). Additionally, MyHC-fast proportions were significantly different between groups (t = 2.38, p = 0.029) (Table 4-3). The fiber type proportions were significantly different in the older group (t - 3.00,p = 0.017) Table 4-3 Fiber Type Morphologic Data Group Fiber Type Fiber CSA (pm2 ) Myonuclei/ Muscle Fiber Fiber Type Proportions Young MyHC-slow 6431± 1859 3.0 ± 0.6 52 ± 13% (n = 10) a(173 ± 24) MyHC-fast 6257± 1721 3.0 ± 0.5 48 ± 13% Older MyHC-slow 5804 ± 1500f 2.6 ± 0.5 66 ± 17%* (n = 9) a(157± 14) MyHC-fast 4714± 1 3 1 0 ft 2.3 ±0.5** 34 ± 17%** a Mean Number o f muscle fibers analyzed. * Significantly greater than Young group MyHC-slow. ** Significantly less than Young group MyHC-fast. t Significantly greater than Older group MyHC-fast F-CSA. f t Significantly less than Young MyHC-fast F-CSA. Skeletal Muscle Fiber Cross-Sectional Area. Skeletal muscle fiber CSA data are presented in Table 4-2. There was a significant between group difference for muscle fiber CSA (F = 9.23, p = 0.007) (Table 4-2). There was no significant within group difference for muscle fiber CSA (F = 0.08,/? = 0.78) and there was no interaction effect (F = 0.18,_p = 0.68). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Independent /-test revealed differences between groups at Pre-Exercise biopsy (t = 2.63, p = 0.018), and Post-Exercise biopsy (t = 2.2A,p = 0.039). When fiber CSA was analyzed by fiber type there was no difference between groups for MyHC-slow (t = 0.90, p = 0.40). However there was as significant between group difference for MyHC-fast fiber CSA (t = 2.39,p = 0.029) (Table 4-3). Additionally, fiber CSA for MyHC-slow was significantly greater than the fiber CSA for MyHC-fast within the older group ( t- 3.18,p = 0.013) (Table 4-3). Intraclass correlation coefficient for fiber CSA performed on three randomly selected subjects analyzed six times was 0.9936. Myonuclei/Muscle Fiber. Data for myonuclei are presented in Table 4-2. There was no between group difference for the number of myonuclei/muscle fiber (F = 2.39, p = 0.14) (Table 4-2). Additionally, there were no significant within group differences for the number of myonuclei/muscle fiber (F = 0.43, p = 0.52), and no interaction effect (F = 1.00, p = 0.33). When myonuclei/muscle fiber was analyzed according to fiber type by Independent /-test there was no significant difference between groups for MyHC-slow (/ = 1.64,/? = 0.12), but there was a significant difference between groups for MyHC-fast (t = 3.29,p = 0.004). When myonuclei/muscle fiber was analyzed according to fiber type there was no significant difference within the young group (t = 0.16,p = 0.88) or the older group (/ = 2.16, p = 0.063) (Table 4-3). Intraclass correlation coefficient for myonuclei/muscle fiber performed on three randomly selected subjects analyzed six times was 0.9936. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Central Myonuclei/Muscle Fiber. There were also no significant between group differences for the number of centrally located myonuclei per muscle fiber (F = 0.42, p = 0.53) (Table 4-2). Additionally, there were no significant within group difference (F = 0.93, p = 0.35) and there was no effect of time (F = 0.1 \,p - 0.75). Satellite Cells. Comparative images for young and older muscle are depicted in Figure 4-3. Additionally, we observed several incidences where the NCAM positive staining included two nuclei in apposition to each other (i.e., two satellite cells) within the same muscle cell. An example from a 65 year old male is provided in Figure 4-4. We believe this to potentially be visual evidence of satellite cell proliferation. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-3 Representative Digital Microphotographs Young (left column) and Older muscle (right column). Top row, NCAM stain; middle row, DAPI stain; and bottom row, Laminin stain with NCAM inset from top row. Arrows indicate location of satellite cells in each image. Image obtained at 40x magnification. Calibration bars = 25 pm 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lam in in + D A P I II Laminin + NCAM li Figure 4-4 Digital Microphotographs of Cell Division. Post-Exercise biopsy obtained from a 65 year old man. Top Image: Merge o f green = Laminin (muscle cell boundary) and blue = DAPI (stains all nuclei). Bottom Image: Merge of green = Laminin and red = NCAM (satellite cells only). Image obtained at 60x magnification. Calibration bars = 25 pm 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Satellite Cells/Muscle Fiber. Values for satellite cells/muscle fiber for individual subject are shown in Figure 4-5, and group means are presented in Table 4-2. There was a significant between group difference for satellite cells/muscle fiber (F = 13.46,/) = 0.002). Similarly, there was a significant difference within groups for satellite cells/muscle fibers (F = 76.03,/? < 0.001) with a significant effect of time (F = 22.96, p < 0.001). Independent /-tests revealed no significant group difference at Pre-Exercise biopsy (baseline) for satellite cells/muscle fiber (/ = 0.75,/? = 0.46), however a significant difference existed at Post-Ex biopsy (/ = 4.58,/? = 0.001). Paired samples /-test showed a significant increase in satellite cells/muscle fiber at Post-Exercise biopsy relative to Pre-Exercise biopsy in both young (/ = 7.90,/? < 0.001) and older (/ = 4.35, p = 0.002) men. The mean change in satellite cells/muscle fiber by independent /-test was significantly greater in magnitude in the young (0.103 ± 1.3) compared to older muscle (0.03 ± 0.02) (/ = 4.79, p < 0.001). Figure 4-6, shows the percent increase form Pre-Exercise biopsy (baseline) for satellite cells/muscle fiber. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.25 0.23 0.21 C/> 3 | 0.19 (D % 0.17 0 ) r o 0.15 C O « 0.13 < D J3 £ 0.11 1 6 p 0.09 ^ 0.07 0.05 Pre Post Figure 4-5 Individual Values for Satellite Cells/Muscle Fiber Black dashed lines = Young Men Grey solid lines = Older Men 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 250 < l > _ Q L l <D - - O < D £ < / > ^ S i o £ C (D - o 5 ? 0 ) CO C O r 200 150 100 50 Young (n=10) Older (n=9) Figure 4-6 Percent Increase in Satellite Cells/Muscle Fiber from Baseline Satellite Cells Proportions. Satellite cell proportions are shown in Table 4-2. There was a significant between group difference for satellite cell proportions (F = 6.85, p = 0.018), and a significant within group difference for satellite cell proportions (F = 85.53,/? < 0.001), and a significant effect of time was observed (F = 14.97, p - 0.001) (Table 4- 2). Independent t-tests revealed no significance between group differences at Pre- Exercise biopsy (baseline) (t = 0.15, p = 0.88); however, a significant difference was observed at Post-Exercise biopsy (t = 3.50,p = 0.003) (Table 4-2). Paired samples t- test showed a significant increase in satellite cell proportions at Post-Exercise biopsy relative to Pre-Exercise biopsy in both young (t = 2.23, p < 0.001) and older (t = 6.61 ,p < 0.001) men. The mean change in satellite cell proportions by independent t-test revealed that the magnitude of change was significantly greater in the young muscle (it = 4.02,/? = 0.002) (Table 4-2). 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The results from this investigation demonstrate a significant increase in satellite cells/muscle fiber and proportions 24 hours after a single bout of maximal- eccentric exercise in both young and older muscle. The magnitude of change, however, was significantly greater in young men. These data suggest that at the 24- hour time point, fewer satellite cells from muscle of older subjects proliferated in response to maximal-eccentric exercise compared to those in the younger subjects. To our knowledge this is the first report on satellite cell proliferation in young and older men at the 24-hour time point. Comparison of our data to other aging studies conducted in humans is difficult due the differences in study design. Kadi et al., (93) observed an increase in satellite cell proportions from trapezius muscle of women who strength-trained for a period of 10 weeks. Roth et al., (142) observed a significant increase in satellite cell numbers at the end of nine weeks of strength training in young and older men and women. The results from those two studies contrast to the results of Hikida et al., (79) who reported no change in satellite cell proportions from the vastus lateralis muscles of young and older men strength-trained for eight and 16 weeks, respectively. Our results support the findings of Kadi et al., (93) and Roth et al., (142) with a proliferative response from satellite cells stimulated by exercise. However, the magnitude of response was different between young and older men in our study, although unlike our findings Roth et al., (142) reported no difference between young and older men after nine weeks of strength training. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Our results are similar to the findings of Hameed et al., (75) who observed a blunted response in young (29.5 ±1.5 years) versus older (74.4 ±1.8 years) men two and a half hours after resistive exercise. That study demonstrated a significantly lower expression levels of MGF mRNA from muscle of older subjects after a unilateral bout of knee extensor exercise consisting of 10 sets of six repetitions at 80% of their 1-RM (75). That study also demonstrated an 80% (non-significant) increase in MyoD expression from muscle of young subjects while the expression in older subjects remained relatively unchanged (75). In a rat model, there was a significant age-related attenuation in MyoD expression (mRNA and protein level) two days after exercise (172). Suggesting that significance may have been reached between the two and a half hour time point of Hameed et al., (75) and the 48-hour endpoint of Tamaki et al., (172). Moreover, a recent report by Conboy et al., (42) demonstrated diminished Delta expression, a ligand for the Notch-1 receptor important for satellite cell activation, from satellite cells of older mice relative to young after muscle injury. That study clearly demonstrated reduced satellite cell proliferation in older animals and provided insight as to the mechanisms involved in the reduced regenerative potential in muscle of older animals (42). Crameri et al., (45) recently reported on satellite cell proliferation in the vastus lateralis muscle of eight male (25 ± 3 years) subjects after eccentric loading. That study demonstrated a significant increase in satellite cells proportions at four (7.57% ± 3.36 exercise leg versus 3.39% ± 0.08 control leg) and eight (6.94% ± 8.27 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. exercise leg versus 3.07% ± 0.89 control leg) days post exercise (45). Tissue was also collected in that study two days post exercise, however, satellite cell proportions (6.38% ± 1.23 exercise leg versus 3.24% ±1.7 control leg) were not significantly elevated at that time point (45). Our satellite cell proportions at 24 hours in young men are very similar to what Crameri et al., (45) report 48 hours after maximal eccentric loading. That study’s subjects performed three exercises each separated by five minutes. The first exercise consisted of 50 single-leg ‘drop down’ jumps from a height of 45 cm, followed by eight sets of 10 maximal-eccentric knee extensions at 30°/second using the KinCom isokinetic dynamometer, which was followed by eight more sets of 10 maximal-eccentric knee extensions this time at 180°/second. Although their 48-hour satellite cell proportions were not significant their satellite cell percentages are similar to what we report in similarly aged men. Interestingly, our maximal-eccentric exercise protocol consisted of a total of 92 repetitions, whereas Crameri et al., (45) incorporated 50 single leg ‘drop down’ and a total of 160 eccentric leg extensions performed at two different isokinetic speeds. That exercise protocol potentially provided a greater muscle damage stimulus for satellite cell activation yet the proliferation percentages between studies appear similar. This may suggest that in young men, satellite cell activation can be achieved with a lower total number of maximal-eccentric repetitions. Similar to the reports from mice (42) which demonstrate no significant change with age in satellite cell proportions, our Pre-Exercise biopsy (baseline) results demonstrate similar satellite cell proportions between young and older men. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These findings are similar to those of Hikida et al., (79) who reported similar satellite cell proportions of 2.4% for young (22.5 ± 5.8 years) and 2.3% for old (65.0 ± 6.0 years), Roth et al., (141), 2.8% for young (25 ± 3 years) and 1.7% for older (69 ± 3 years) men, Crameri et al., (45), 2.59% for young (25 ± 3 years) men, and Charifi et al., (37) who reported satellite cell proportions of 2.4% in older men (73 ± 3 years) at baseline. However, Kadi et al., (89) reported significantly higher proportions of satellite cells from young men (25 ± 3 years) relative to older men (69 ± 3) (7.1% versus 4.4%, respectively). The apparent discrepancies between studies may be due to the muscles sampled. Our data as well as those reporting similar values mentioned above (37, 45, 79, 142) were derived from biopsies of the vastus lateralis muscle, whereas Kadi et al., (89) sampled tissue from the tibialis anterior (TA) muscle. Paradoxically, animal studies suggest a greater proportion (5-6 times) of satellite cells in slow oxidative (Type I) muscle fibers compared with glycolytic fibers (Type II) (149), which in the predominantly glycolytic TA muscle should result in reduced satellite cell percentages relative to heterogeneous vastus lateralis muscle. The apparent discrepancies may be due to species variability or a true difference in satellite cell proportions between muscles in humans. Taken together these findings indicate that the number of satellite cells does not decline in older men, which is different from what occurs in the rat (68, 166) from which the majority of the early data on satellite cells was obtained. There were several limitations of this study. Although testosterone levels were similar in the two study groups (possibly due to small number of subjects), 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. testosterone levels in the older subjects are generally lower than in the younger subjects (76, 122). Whether differences in endogenous testosterone levels affect satellite cell activation during a bout of maximal-eccentric exercise is unknown, but evidence indicates that exogenous testosterone activates satellite cells (163). Similarly, the growth hormone-IGF-I axis declines with aging and IGF-I is critical for satellite cell division and development of new myotubes (2). Whether age- related declines in systemic levels of growth hormone or IGF-I (not measured in this study) influenced the local effects of IGF-I in skeletal muscle during exercise is unknown, although this is of concern since systemic administration of either GH or IGF-I increases local myofibrillar protein synthesis (64, 65). The lower hemoglobin and albumin in the older subjects is consistent with a chronic inflammation typical of the aging process, which could augment the catabolic response in muscle. Whether inflammation affects the satellite response to maximal-eccentric exercise is unknown. In addition, we did not carefully control for recreational exercise or regular training but doubt that lower levels of such exertion compared to the maximum bouts of eccentric exercise tested in this study would have substantively affected the results. We were unable to account for any potential activation of satellite caused by inflammation resulting from the initial biopsy procedure as we did not have a non-exercise control group (i.e., subjects who only underwent the two biopsies). However, any potential influences of muscle damage or inflammation caused by the Pre-Exercise biopsy is likely to have been minimal and potentially would have affected the groups equally as there were no differences between groups 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for the number of days between biopsies. Moreover, satellite cell proportions obtained from biopsies collected from the control leg by Crameri et al., (45) were not significantly different and those biopsies were obtained approximately one cm apart. In conclusion, our data suggest that proliferation of satellite cells occurs early in skeletal muscle (within 24 hours) in response to maximal-eccentric exercise in both young and older men. Indirectly these results support a satellite cell cycle time approximating 24 hours. Lastly, our data support a role for diminished satellite cell activation as a potential source of decreased regenerative and/or hypertrophic capacity in older muscle which may partially explain the difference in the anabolic response demonstrated at the end of strength training programs in older adults (73, 183). 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V SUMMARY AND CONCLUSIONS Summary of Study I The purpose of this study was to determine the temporal effects of continuous supraphysiologic levels of TP on satellite cell proliferation in the diaphragm muscle of sedentary gonadally intact male rats. The diaphragm was chosen to reduce the potentially confounding effect of testosterone-induced modulation of locomotor activity. This was achieved by determining the effects of 0 (Sham-Control), 7,14, and 28 days of supraphysiologic testosterone propionate on satellite cell proliferation in the gonadally-intact male rat diaphragm. Pathogen-free animals were randomly assigned to two groups, sham-operated controls or those treated with TP. Continuous release capsules were made from Silastic™ tubing, checked for leakage and pre equilibrated in sterile saline for 24 hours prior to implantation. Sham- controls (n = 5) were subcutaneously implanted with a single empty 45-mm capsule. Male rats receiving testosterone treatment were subcutaneously implanted with two 45-mm capsules filled with TP (TP; 4-androsten-17p -ol-3-one 17 propionate; Steraloids) under ketamine:xylazine anesthesia. Similar methods have been used previously by us and have shown that serum testosterone levels are elevated approximately 6.5 times greater than physiologic conditions after 28 days of testosterone treatment. Immunohistochemical analysis of the diaphragm tissue cross-sections from the gonadally intact male rat revealed the following results: 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. Diaphragm muscle fiber CSA for all muscle fiber types did not change with TP treatment. 2. The fiber CSA for diaphragm muscle fibers expressing the MyHC-slow isoform did not change with TP treatment. 3. The number of myonuclei/muscle fiber remained stable between groups. 4. The myonuclear domain [pm /myonuclei] also did not vary with TP treatment duration within the 28 days. 5. Satellite cells/muscle fiber increased in the rat diaphragm muscle after seven and 14 days of TP treatment. 6. Satellite cells/muscle fiber returned to baseline by 28 days despite continued TP treatment in the rat diaphragm muscle. 7. Satellite cell proportions [satellite cell/(satellite cells + myonuclei)] x 100, increased in the rat diaphragm muscle after seven and 14 days of TP treatment. 8. Satellite cell proportions returned to baseline values by day 28 of the study despite continued supraphysiologic TP treatment. These findings suggest that supraphysiologic administration of TP stimulates satellite cells to proliferate significantly by seven days in the male rat diaphragm muscle. The expansion of satellite cell numbers remains significantly elevated until day-14. By day-28 satellite cells/muscle fiber and proportions return to baseline. However, these satellite cell stimulated to proliferate did not differentiate and become new myonuclei unless myonuclear turnover increased in proportion to the proliferation 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. satellite cell. Similarly to myonuclear numbers the fiber CSA of the diaphragm muscle fibers as well as a subset of muscle cells expressing the MyHC-slow isoform did not increase with TP administration. Study one tested the hypothesis that satellite cells would expand in number in proportion to the duration of TP treatment. This hypothesis was false; satellite cell proliferation due to supraphysiologic AAS was temporal. Summary of Study II The goal of this study was to determine the amount of satellite cell proliferation in young and older muscle 24 hours after a single bout of unilateral maximal- eccentric exercise. Ten young (28 ± 5 years) and nine older (68 ± 6 years) men were found to be eligible and were enrolled in the study. Each subject had two biopsies performed on the dominant vastus lateralis muscle separated in time by (12 ± 3 and 12 ± 1 days) for young and older men, respectively. The second biopsy was preceded in time (24 ±0.5 and 24 ± 0.4 hours) for young and older men, respectively by a single bout of maximal-eccentric exercise of the dominant quadriceps muscle group consisting of one set of 12 repetitions and five sets of 16 repetitions. Immunohistochemical analysis of the vastus lateralis tissue cross-sections from each age group revealed the following results: 1. The average peak torque (Nm) for sets 1 & 2 and 3 & 4 were significantly greater in the young men. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. The young had significantly greater proportions of fast fiber types. 3. The older subjects had significantly greater proportions of slow fiber types. 4. The fiber CSA from all fibers regardless of fiber type was significantly greater in the muscles from young subjects. 5. The fiber CSA did not change between biopsies in young or older men. 6. The fiber CSA for slow and fast fibers in young subjects were not significantly different. 7. The fiber CSA for slow fibers was significantly greater than the fiber CSA of fast fibers of older subjects. 8. The fiber CSA measurements by fiber type between biopsies did not change in either age group. 9. The number of myonuclei/muscle fiber was not significantly different between groups. 10. The number of myonuclei/muscle fibers was not significantly different between Pre- and Post-Exercise biopsies. 11. When separated according to fiber type, no significant different for myonuclei/muscle fiber was observed for the young group. 12. Myonuclei/slow muscle fibers were significantly greater than myonuclei/fast muscle fibers for the older group. 13. There was no significant difference between groups for the number of centrally located nuclei/muscle fiber. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14. There was no significant difference for either age group for centrally located nuclei/muscle fiber between Pre-and Post-Exercise biopsy. 15. Satellite cells/muscle fiber was not significantly different between age groups at baseline. 16. Satellite cells/muscle fiber increased significantly in both age groups 24 hours after a single bout of maximal-eccentric exercise. 17. The magnitude of change for satellite cells/muscle fiber was significantly greater in the muscles of young men. 18. Satellite cell proportions were not significantly different between age groups at baseline. 19. Satellite cell proportions increased significantly in both age groups 24 hours after a single bout of maximal-eccentric exercise. 20. The magnitude of change for satellite cell proportions was significantly greater in the muscles of young men. From these results we conclude that the repeatability of immunohistochemical methodology used in this study was demonstrated by comparisons of Pre-and Post- Exercise biopsy samples for variables of fiber CSA and myonuclei/muscle fiber. Repeatability of immunohistochemical methodology was established when comparing the fiber CSA by fiber type and myonuclei/muscle fiber type. We report immunohistochemical methodological validity as baseline measures (fiber CSA, myonuclei/muscle fiber, satellite cells/muscle fiber, and satellite cell proportions) were similar to published reports for both age groups. We also report 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. validity of intervention stimulus for young subjects based on the similarity of satellite cell proportional increases between studies. Study two tested the hypothesis that satellite cell numbers would increase in young and older men 24 hours after a single bout of maximal-eccentric exercise. The research hypothesis was accepted as tenable. We also tested the hypothesis that the magnitude of change, if observed, would be less in older men. This hypothesis was statistically supported. These results suggest that the satellite cells residing within the basal lamina of skeletal muscle and positively expressing NCAM expand in response to a single bout of maximal-eccentric exercise totaling 92 repetitions. Moreover, this exercise stimulus did not result in significant discomfort as reported by the rating of discomfort scale. This mode of exercise was well tolerated by 18 out of 19 subjects and thus may potentially be used therapeutically for satellite cell activation. Varying levels of cytokines, growth factors, endogenous or exogenous steroid hormone or other anabolic agents can potentially effect either independently or interdependently the overall activation, proliferation or differentiation of the skeletal muscle satellite cell pool. We report that TP is able to increase the number of M- cadherin positive cells. Current research directed towards determining the mechanism(s) responsible for the age-related attenuation of satellite cell proliferation is needed. Potential contributors may exert independent or interdependent control over myogenic response from skeletal muscle cells and the contribution of each factor may change with age. We report that 92 maximal-eccentric contractions is 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. able to increase the number of NCAM positive satellite cells within 24 hours. This dissertation has contributed to the satellite cell literature by demonstrating a temporal response of satellite cells to supraphysiologic levels of circulating testosterone and by showing that age affects the ability of satellite cells to respond to a single bout of maximal-eccentric loading 24 hours later. Mathematically, the potential contribution of diminished satellite cell apoptosis to the increases observed in either study is remote, at best. Estimated turnover of myonuclei of 1-2% suggest a slow renewal rate. Thus, the expansion of satellite cells observed far exceeds the potential increase in satellite cell numbers that would result from halting apoptosis. A 2% increase in the number of satellite cells per week would not account for the approximately 3-fold increase within seven, and nearly 4-fold increase at 14 days of TP treatment observed in the rat study. Moreover, the 24 hours between the maximal-eccentric exercise and the Post exercise biopsy could not have resulted in the 150 and 50% increase in satellite cell numbers in young and older groups, respectively. It remains that, the most plausible explanation for the expansion of satellite cells observed in the two studies resulted from increased cell division and not from attenuated apoptosis. Future Directions What remains to be determined is the activation of satellite cells in humans placed on a progressive resistance training program and supplemented with supraphysiologic levels of testosterone. Moreover, these studies would need to 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. establish the timeline for optimal satellite cell proliferation with testosterone delivery. Additionally, future research is needed to delineate the contribution of MDSC and muscle SPSC that reside within skeletal muscle. The prevailing thought today is that satellite cells residing within the basal lamina constitute 99% of the muscle precursor pool under normal conditions of repair and mass accretion. The future for human skeletal muscle satellite cell research is ripe. Strategies to augment skeletal muscle mass invariably must involve the satellite cell and that research is in its infancy. Moreover, hypothesis based mechanistic research must be conducted in order to uncover potential areas for therapeutic intervention. Our current knowledge on testosterone is incomplete and will require the marriage of animal experimentation and clinical trials. In the interest of pragmatism, anabolic steroid/growth hormone treatment will achieve the greatest results and demands the most attention in terms of sarcopenia-based intervention. Invariably the path towards muscle mass maintenance and accretion will involve nutrition, endocrine therapy, and exercise (anaerobic and anabolic), which must be monitored to elucidate the interactions of these singularly anabolic treatments with testosterone. Power estimates for future studies in the human model based on these data are presented in APPENDIX H. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1. Adams GR. Invited Review: Autocrine/paracrine IGF-I and skeletal muscle adaptation. JAppl Physiol 93: 1159-1167, 2002. 2. Adams GR. 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. 3. Adams GR and Haddad F. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. JAppl Physiol 81:2509-2516, 1996. 4. Adams GR, Haddad F, and Baldwin KM. Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. JAppl Physiol 87: 1705-1712,1999. 5. Allen DL, Roy RR, and Edgerton VR. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22: 1350-1360, 1999. 6. Allen RE and Boxhorn LK. Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta. J Cell Physiol 133: 567- 572, 1987. 7. Alway SE, Carson JA, and Roman WJ. Adaptation in myosin expression of avian skeletal muscle after weighting and unweighting. J Muscle Res Cell Motil 16: 111-122,1995. 8. Alway SE, Grumbt WH, Gonyea WJ, and Stray-Gundersen J. Contrasts in muscle and myofibers of elite male and female bodybuilders. JAppl Physiol 67:24-31, 1989. 9. Armand O, Boutineau AM, Mauger A, Pautou MP, and Kieny M. Origin of satellite cells in avian skeletal muscles. Arch Anat Microsc Morphol Exp 72:163-181, 1983. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10. Armstrong RB, Ogilvie RW, and Schwane JA. Eccentric exercise-induced injury to rat skeletal muscle. JAppl Physiol 54: 80-93, 1983. 11. Asakura A, Seale P, Girgis-Gabardo A, and Rudnicki MA. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159: 123- 134,2002. 12. Bartsch W, Krieg, M., Voigt, K.D. Quantification of endogenous testosterone, 5alpha-dihhydorotestosterone and 5alpha-androstane-3alpha, 17beta-diol in subcellular fractions of he prostate, bulbocaemosus/levator ani muscle, skeletal muscle and heart muscle of the rat. Journal o f Steroid Biochemistry 13: 259-264, 1980. 13. Bergstrom J. Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. ScandJ Clin Lab Invest 35: 609-616, 1975. 14. Bhasin S. Testosterone supplementation for aging-associated sarcopenia. J Gerontol A Biol Sci Med Sci 58: 1002-1008, 2003. 15. Bhasin S, Storer TW, Asbel-Sethi N, Kilbourne A, Hays R, Sinha-Hikim I, Shen R, Arver S, and Beall G. Effects of testosterone replacement with a nongenital, transdermal system, Androderm, in human immunodeficiency virus-infected men with low testosterone levels. J Clin Endocrinol Metab 83: 3155-3162, 1998. 16. Bhasin S, Storer TW, Berman N, Yarasheski KE, Clevenger B, Phillips J, Lee WP, Bunnell TJ, and Casaburi R. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab 82: 407-413, 1997. 17. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, and Storer TW. Testosterone dose- response relationships in healthy young men. Am J Physiol Endocrinol Metab 281: El 172-1181, 2001. 18. Bhasin S, Woodhouse L, and Storer TW. Proof of the effect of testosterone on skeletal muscle. J Endocrinol 170: 27-38, 2001. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19. Bischoff R. Cell cycle commitment of rat muscle satellite cells. J Cell Biol 111: 201-207, 1990. 20. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 208: 505- 515, 1997. 21. Bischoff R. Interaction between satellite cells and skeletal muscle fibers. Development 109: 943-952, 1990. 22. Bischoff R. Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115: 129-139, 1986. 23. Bischoff R. The satellite cell and muscle regeneration. New York: McGraw- Hill, 1994. 24. Bischoff R and Franzini-Armstrong C. Satellite and Stem Cells and Muscle Regeneration. New York: McGraw-Hill, Inc., 2004. 25. Bisschop A, Gayan-Ramirez G, RoIIier H, Dekhuijzen PN, Dom R, de Bock Y, and Decramer M. Effects of nandrolone decanoate on respiratory and peripheral muscles in male and female rats. JAppl Physiol 82: 1112- 1118,1997. 26. Blanco CE, Popper P, and Micevych P. Anabolic-androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat. Neuroscience 78: 873-882, 1997. 27. Blanco CE, Zhan WZ, Fang YH, and Sieck GC. Exogenous testosterone treatment decreases diaphragm neuromuscular transmission failure in male rats. JAppl Physiol 90: 850-856, 2001. 28. Blough ER and Linderman JK. Lack of skeletal muscle hypertrophy in very aged male Fischer 344 x Brown Norway rats. JAppl Physiol 88: 1265- 1270, 2000. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29. Burton K, Zagotta WN, and Baskin RJ. Sarcomere length behaviour along single frog muscle fibres at different lengths during isometric tetani. J Muscle Res Cell Motil 10: 67-84, 1989. 30. Cameron-Smith D. Exercise and skeletal muscle gene expression. Clin Exp Pharmacol Physiol 29: 209-213, 2002. 31. Cannon JG, Meydani SN, Fielding RA, Fiatarone MA, Meydani M, Farhangmehr M, Orencole SF, Blumberg JB, and Evans WJ. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol 260: R1235-1240,1991. 32. Cannon JG, Orencole SF, Fielding RA, Meydani M, Meydani SN, Fiatarone MA, Blumberg JB, and Evans WJ. Acute phase response in exercise: interaction of age and vitamin E on neutrophils and muscle enzyme release. Am J Physiol 259: R1214-1219, 1990. 33. Cao B and Huard J. Muscle-derived stem cells. Cell Cycle 3: 104-107, 2004. 34. Carson JA and Alway SE. Stretch overload-induced satellite cell activation in slow tonic muscle from adult and aged Japanese quail. Am J Physiol 270: C578-584, 1996. 35. Carson JA, Alway SE, and Yamaguchi M. Time course of hypertrophic adaptations of the anterior latissimus dorsi muscle to stretch overload in aged Japanese quail. J Gerontol A Biol Sci Med Sci 50: B391-398, 1995. 36. Charge SB and Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 84: 209-238, 2004. 37. Charifi N, Kadi F, Feasson L, and Denis C. Effects of endurance training on satellite cell frequency in skeletal muscle of old men. Muscle Nerve 28: 87-92, 2003. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38. Cheek DB, Holt AB, Hill DE, and Talbert JL. Skeletal muscle cell mass and growth: the concept of the deoxyribonucleic unit. Pediat Res 5: 312-328, 1971. 39. Clark AS and Henderson LP. Behavioral and physiological responses to anabolic-androgenic steroids. Neurosci Biobehav Rev 27: 413-436, 2003. 40. Colliander EB and Tesch PA. Effects of eccentric and concentric muscle actions in resistance training. Acta Physiol Scand 140: 31-39, 1990. 41. Colliander EB and Tesch PA. Responses to eccentric and concentric resistance training in females and males. Acta Physiol Scand 141: 149-156, 1991. 42. Conboy IM, Conboy MJ, Smythe GM, and Rando TA. Notch-mediated restoration of regenerative potential to aged muscle. Science 302: 1575-1577, 2003. 43. Cornelison DD, Olwin BB, Rudnicki MA, and Wold BJ. MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient.PG - 122-37. Dev Biol 224, 2000. 44. Cornelison DD and Wold BJ. Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270-283, 1997. 45. Crameri RM, Langberg H, Magnusson P, Jensen CH, Schroder HD, Olesen JL, Suetta C, Teisner B, and Kjaer M. Changes in satellite cells in human skeletal muscle after a single bout of high intensity exercise. J Physiol 558: 333-340, 2004. 46. Crenshaw AG, Thornell LE, and Friden J. Intramuscular pressure, torque and swelling for the exercise-induced sore vastus lateralis muscle. Acta Physiol Scand 152: 265-277, 1994. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47. Danneskiold-Samsoe B, Kofod V, Munter J, Grimby G, Schnohr P, and Jensen G. Muscle strength and functional capacity in 78-81-year-old men and women. Eur JAppl Physiol Occup Physiol 52: 310-314, 1984. 48. De Angelis L, Berghella L, Coletta M, Lattanzi L, Zanchi M, Cusella-De Angelis MG, Ponzetto C, and Cossu G. Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J Cell Biol 147: 869-878, 1999. 49. Decary S, Mouly V, Hamida CB, Sautet A, Barbet JP, and Butler- Browne GS. Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy. Hum Gene Ther 8: 1429-1438, 1997. 50. Dedkov El, Borisov AB, Wernig A, and Carlson BM. Aging of skeletal muscle does not affect the response of satellite cells to denervation. J Histochem Cytochem 51: 853-863, 2003. 51. Doumit ME, Cook DR, and Merkel RA. Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology 137: 1385-1394, 1996. 52. Dreyer HC, Hawkins SA, Schroeder ET, and Wiswell RA. Muscle quality in master athletes drops significantly after age 65. Med Sci Sports Exerc 34: S98, 2002. 53. Dudley GA, Tesch PA, Miller BJ, and Buchanan P. Importance of eccentric actions in performance adaptations to resistance training. Aviat Space Environ Med 62: 543-550, 1991. 54. Ecob-Prince MS, Jenkison M, Butler-Browne GS, and Whalen RG. Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system . J Cell Biol 103: 995-1005,1986. 55. Fano G, Di Tano G, Parabita M, Beltramin A, and Mariggio M. Stem Cells in Adult Skeletal Muscle Tissue: More than a Working Hypothesis. Basic Appl Myol 14: 13-15, 2004. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56. Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J, Achacosa A, Lieberman SA, Tipton K, Wolfe RR, and Urban RJ. Testosterone administration to older men improves muscle function: molecular and physiological mechanisms. Am J Physiol Endocrinol Metab 282: E601-607, 2002. 57. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, and Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279: 1528-1530, 1998. 58. Fiatarone MA and Evans WJ. The etiology and reversibility of muscle dysfunction in the aged. J Gerontol 48 Spec No: 77-83, 1993. 59. Fiatarone MA, Marks EC, Ryan ND, Meredith CN, Lipsitz LA, and Evans WJ. High-intensity strength training in nonagenarians. Effects on skeletal muscle. Jama 263: 3029-3034, 1990. 60. Friden J and Lieber RL. Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components. Acta Physiol Scand 171: 321-326, 2001. 61. Friden J and Lieber RL. Segmental muscle fiber lesions after repetitive eccentric contractions. Cell Tissue Res 293: 165-171, 1998. 62. Friden J and Lieber RL. Structural and mechanical basis of exercise- induced muscle injury. Med Sci Sports Exerc 24: 521-530, 1992. 63. Friden J, Sjostrom M, and Ekblom B. Myofibrillar damage following intense eccentric exercise in man. IntJSports Med 4: 170-176, 1983. 64. Fryburg DA, Gelfand RA, and Barrett EJ. Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. Am J Physiol 260: E499-504, 1991. 65. Fryburg DA, Jahn LA, Hill SA, Oliveras DM, and Barrett EJ. Insulin and insulin-like growth factor-I enhance human skeletal muscle protein 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. anabolism during hyperaminoacidemia by different mechanisms. J Clin Invest 96: 1722-1729, 1995. 66. Gayan-Ramirez G, Rollier H, Vanderhoydonc F, Verhoeven G, Gosselink R, and Decramer M. Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm. JAppl Physiol 88: 26-34, 2000. 67. Gibala MJ, MacDougall JD, Tarnopolsky MA, Stauber WT, and Elorriaga A. Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise. J Appl Physiol 78: 702-708,1995. 68. Gibson MC and Schultz E. Age-related differences in absolute numbers of skeletal muscle satellite cells. Muscle Nerve 6: 574-580, 1983. 69. Gibson MC and Schultz E. The distribution of satellite cells and their relationship to specific fiber types in soleus and extensor digitorum longus muscles. Anat Rec 202: 329-337, 1982. 70. Greig CA, Young A, Skelton DA, Pippet E, Butler FM, and Mahmud SM. Exercise studies with elderly volunteers. Age Ageing 23: 185-189, 1994. 71. Grounds MD. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann N Y Acad Sci 854: 78-91, 1998. 72. Grounds MD, White JD, Rosenthal N, and Bogoyevitch MA. The role of stem cells in skeletal and cardiac muscle repair. JHistochem Cytochem 50: 589-610, 2002. 73. Hakkinen K, Kallinen M, Izquierdo M, Jokelainen K, Lassila H, Malkia E, Kraemer WJ, Newton RU, and Alen M. Changes in agonist-antagonist EMG, muscle CSA, and force during strength training in middle-aged and older people. J Appl Physiol 84: 1341-1349, 1998. 74. Hall ZW and Ralston E. Nuclear domains in muscle cells. Cell 59: 771-772, 1989. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75. Hameed M, Orrell RW, Cobbold M, Goldspink G, and Harridge SD. Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise. J Physiol 547: 247-254, 2003. 76. Harman SM, Metter EJ, Tobin JD, Pearson J, and Blackman MR. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab 86: 724-731,2001. 77. Hasten DL, Pak-Loduca J, Obert KA, and Yarasheski KE. Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in 78-84 and 23-32 yr olds. Am J Physiol Endocrinol Metab 278: E620-626, 2000. 78. Hawke TJ and Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91: 534-551, 2001. 79. Hikida RS, Eriksson A, Holmner S, and Thornell LE. Is hypertrophy limited in elderly muscle fibers? A comparison of elderly and young strength- trained men. Basic Appl Myol 8: 419-427, 1998. 80. Holmes GM and Sachs BD. Erectile function and bulbospongiosus EMG activity in estrogen-maintained castrated rats vary with behavioral context. Horm Behav 26: 406-419, 1992. 81. Huard J, Cao B, and Qu-Petersen Z. Muscle-derived stem cells: potential for muscle regeneration. Birth Defects Res Part C Embryo Today 69: 230- 237, 2003. 82. Hurme T and Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exerc 24: 197-205, 1992. 83. Ilia I, Leon-Monzon M, and Dalakas MC. Regenerating and denervated human muscle fibers and satellite cells express neural cell adhesion molecule recognized by monoclonal antibodies to natural killer cells. Ann Neurol 31: 46-52, 1992. 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84. Jacobs SC, Wokke JH, Bar PR, and Bootsma AL. Satellite cell activation after muscle damage in young and adult rats. Anat Rec 242: 329-336, 1995. 85. Joubert Y and Tobin C. Satellite cell proliferation and increase in the number of myonuclei induced by testosterone in the levator ani muscle of the adult female rat. Dev Biol 131: 550-557, 1989. 86. Joubert Y and Tobin C. Testosterone treatment results in quiescent satellite cells being activated and recruited into cell cycle in rat levator ani muscle. Dev Biol 169: 286-294, 1995. 87. Joubert Y, Tobin C, and Lebart MC. Testosterone-induced masculinization of the rat levator ani muscle during puberty. Dev Biol 162: 104-110, 1994. 88. Kadi F. Adaptation of human skeletal muscle to training and anabolic steroids. Acta Physiol Scand Suppl 646: 1-52, 2000. 89. Kadi F, Charifi N, Denis C, and Lexell J. Satellite cells and myonuclei in young and elderly women and men. Muscle Nerve 29: 120-127, 2004. 90. Kadi F, Eriksson A, Holmner S, Butler-Browne GS, and Thornell LE. Cellular adaptation of the trapezius muscle in strength-trained athletes. Histochem Cell Biol 111: 189-195, 1999. 91. Kadi F, Eriksson A, Holmner S, and Thornell LE. Effects of anabolic steroids on the muscle cells of strength-trained athletes. Med Sci Sports Exerc 31: 1528-1534, 1999. 92. Kadi F, Schjerling P, Andersen LL, Charifi N, Madsen JL, Christensen LR, and Andersen JL. The effects of heavy resistance training and detraining on satellite cells in human skeletal muscles. J Physiol, 2004. 93. Kadi F and Thornell LE. Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training. Histochem Cell Biol 113: 99-103, 2000. 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94. Kannus P, Jozsa L, Jarvinen TL, Kvist M, Vieno T, Jarvinen TA, Natri A, and Jarvinen M. Free mobilization and low- to high-intensity exercise in immobilization-induced muscle atrophy. J Appl Physiol 84: 1418-1424, 1998. 95. Keliermayer MS and Granzier HL. Elastic properties of single titin molecules made visible through fluorescent F-actin binding. Biochem Biophys Res Commun 221: 491-497, 1996. 96. Keliermayer MS, Smith S, Bustamante C, and Granzier HL. Mechanical manipulation of single titin molecules with laser tweezers. Adv Exp Med Biol 481: 111-126; discussion 127-118,2000. 97. Komulainen J, Koskinen SO, Kalliokoski R, Takala TE, and Vihko V. Gender differences in skeletal muscle fibre damage after eccentrically biased downhill running in rats. Acta Physiol Scand 165: 57-63, 1999. 98. Lamberts SW, van den Beld AW, and van der Lely AJ. The endocrinology of aging. Science 278: 419-424, 1997. 99. Lanier LL, Testi R, Bindl J, and Phillips JH. Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J Exp Med 169:2233-2238, 1989. 100. Larsson L, Grimby G, and Karlsson J. Muscle strength and speed of movement in relation to age and muscle morphology. JAppl Physiol 46: 451- 456, 1979. 101. Lieber RL and Friden J. Selective damage of fast glycolytic muscle fibres with eccentric contraction of the rabbit tibialis anterior. Acta Physiol Scand 133:587-588, 1988. 102. Lieber RL, Shah S, and Friden J. Cytoskeletal disruption after eccentric contraction-induced muscle injury. Clin Orthop: S90-99, 2002. 103. Lieber RL, Thornell LE, and Friden J. Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction. JAppl Physiol 80: 278-284, 1996. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 104. Lindle RS, Metter EJ, Lynch NA, Fleg JL, Fozard JL, Tobin J, Roy TA, and Hurley BF. Age and gender comparisons of muscle strength in 654 women and men aged 20-93 yr. J Appl Physiol 83: 1581-1587, 1997. 105. Link D, Winnekendonk D, Kaufmann U, Palinkas J, Heuser C, Kammann M, and Starzinski-Powitz A. Intercellular Adhesion in Developing and Adult Skeletal Muscle: Analysis of M-Cadherin. Basic Appl Myol 8: 315-323,2004. 106. Lodish H, Berk A, Zipursky SH, Matsudaira P, Baltimore D, and Darnell J. Molecular Cell Biology. Freeman press, 2000. 107. Lowe DA and Alway SE. Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation. Cell Tissue Res 296: 531-539, 1999. 108. Lowe DA, Lund T, and Alway SE. Hypertrophy-stimulated myogenic regulatory factor mRNA increases are attenuated in fast muscle of aged quails. Am J Physiol 275: C155-162, 1998. 109. Lynch NA, Metter EJ, Lindle RS, Fozard JL, Tobin JD, Roy TA, Fleg JL, and Hurley BF. Muscle quality. I. Age-associated differences between arm and leg muscle groups. J Appl Physiol 86: 188-194, 1999. 110. MacDougall JD, Gibala MJ, Tarnopolsky MA, MacDonald JR, Interisano SA, and Yarasheski KE. The time course for elevated muscle protein synthesis following heavy resistance exercise. Can JAppl Physiol 20: 480-486, 1995. 111. Maier F and Bornemann A. Comparison of the muscle fiber diameter and satellite cell frequency in human muscle biopsies. Muscle Nerve 22: 578-583, 1999. 112. Malm C, Nyberg P, Engstrom M, Sjodin B, Lenkei R, Ekblom B, and Lundberg I. Immunological changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies. J Physiol 529 Pt 1: 243-262, 2000. 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 113. Marsh DR, Criswell DS, Carson JA, and Booth FW. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J Appl Physiol S3: 1270-1275, 1997. 114. Maughan RJ, Watson JS, and Weir J. Strength and cross-sectional area of human skeletal muscle. J Physiol 338: 37-49, 1983. 115. Mauro A. Satellite cell of skeletal muscle fibers. JBiophys Biochem Cytol 9: 493-495, 1961. 116. McDonagh MJ and Davies CT. Adaptive response of mammalian skeletal muscle to exercise with high loads. Eur JAppl Physiol Occup Physiol 52: 139-155, 1984. 117. Metter EJ, Lynch N, Conwit R, Lindle R, Tobin J, and Hurley B. Muscle quality and age: cross-sectional and longitudinal comparisons. J Gerontol A Biol Sci Med Sci 54: B207-218, 1999. 118. Miller KJ, Thaloor D, Matteson S, and Pavlath GK. Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle. Am J Physiol Cell Physiol 278: C174-181, 2000. 119. Monks DA, O’Bryant EL, and Jordan CL. Androgen receptor immunoreactivity in skeletal muscle: enrichment at the neuromuscular junction. J Comp Neurol 473: 59-72, 2004. 120. Morgan JE and Partridge TA. Muscle satellite cells. IntJBiochem Cell Biol 35: 1151-1156, 2003. 121. Morley JE, Baumgartner RN, Roubenoff R, Mayer J, and Nair KS. Sarcopenia. J Lab Clin Med 137: 231-243, 2001. 122. Morley JE, Kaiser FE, Perry HM, 3rd, Patrick P, Morley PM, Stauber PM, Vellas B, Baumgartner RN, and Garry PJ. Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metabolism 46: 410-413, 1997. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 123. Moss FP and Leblond CP. Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 170: 421-435, 1971. 124. Nnodim JO. Quantitative study of the effects of denervation and castration on the levator ani muscle of the rat. Anat Rec 255: 324-333, 1999. 125. Nnodim JO. Satellite cell numbers in senile rat levator ani muscle. Mech Ageing Dev 112: 99-111, 2000. 126. Nnodim JO. Testosterone mediates satellite cell activation in denervated rat levator ani muscle. Anat Rec 263: 19-24, 2001. 127. Owino V, Yang SY, and Goldspink G. Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload. FEBS Lett 505: 259-263, 2001 . 128. Partridge TA. Cells that participate in regeneration of skeletal muscle. Gene Ther 9: 752-753, 2002. 129. Phelan JN and Gonyea WJ. Effect of radiation on satellite cell activity and protein expression in overloaded mammalian skeletal muscle. Anat Rec 247: 179-188, 1997. 130. Phillips SM, Tipton KD, Aarsland A, Wolf SE, and Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol 273: E99-107, 1997. 131. Proske U and Morgan DL. Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J Physiol 537: 333-345, 2001. 132. Rasmussen BB and Phillips SM. Contractile and nutritional regulation of human muscle growth. Exerc Sport Sci Rev 31: 127-131, 2003. 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 133. Reimann J, Brimah K, Schroder R, Wernig A, Beauchamp JR, and Partridge TA. Pax7 distribution in human skeletal muscle biopsies and myogenic tissue cultures. Cell Tissue Res 315: 233-242, 2004. 134. Renault V, Rolland E, Thornell LE, Mouly V, and Butler-Browne G. Distribution of satellite cells in the human vastus lateralis muscle during aging. Exp Gerontol 37: 1513-1514, 2002. 135. Rosenblatt JD and Parry DJ. Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. JAppl Physiol 73: 2538-2543, 1992. 136. Rosenblatt JD and Parry DJ. Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle.PG - 2538-43. J Appl Physiol 73,1992. 137. Rosenblatt JD, Parry D J, and Partridge TA. Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60: 39-45, 1996. 138. Rosenblatt JD, Yong D, and Parry DJ. Satellite cell activity is required for hypertrophy of overloaded adult rat muscle. Muscle Nerve 17: 608-613,1994. 139. Roth SM, Ivey FM, Martel GF, Lemmer JT, Hurlbut DE, Siegel EL, Metter EJ, Fleg JL, Fozard JL, Kostek MC, Wernick DM, and Hurley BF. Muscle size responses to strength training in young and older men and women. J Am Geriatr Soc 49: 1428-1433, 2001. 140. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, and Rogers MA. High-volume, heavy-resistance strength training and muscle damage in young and older women. JAppl Physiol 88: 1112-1118, 2000. 141. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, and Rogers MA. Skeletal muscle satellite cell populations in healthy young and older men and women. Anat Rec 260: 351-358, 2000. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. Roth SM, Martel GF, Ivey FM, Lemmer JT, Tracy BL, Metter EJ, Hurley BF, and Rogers MA. Skeletal muscle satellite cell characteristics in young and older men and women after heavy resistance strength training. J Gerontol A Biol Sci Med Sci 56: B240-247, 2001. Roubenoff R. Sarcopenia: effects on body composition and function. J Gerontol A Biol Sci Med Sci 58: M1012-1017, 2003. Roubenoff R and Castaneda C. Sarcopenia-understanding the dynamics of aging muscle. Jama 286: 1230-1231, 2001. Roy RR, Monke SR, Allen DL, and Edgerton VR. Modulation of myonuclear number in functionally overloaded and exercised rat plantaris fibers. JAppl Physiol 87: 634-642,1999. Roy RR, Zhong H, Talmadge RJ, Bodine SC, Fanton JW, Koslovskaya I, and Edgerton VR. Size and myonuclear domains in Rhesus soleus muscle fibers: short-term spaceflight. JGravit Physiol 8: 49-56, 2001. Sabourin LA and Rudnicki MA. The molecular regulation of myogenesis. Clin Genet 57: 16-25, 2000. Sajko S, Kubinova L, Cvetko E, Kreft M, Wernig A, and Erzen I. Frequency of M-Cadherin-stained Satellite Cells Declines in Human Muscles During Aging. J Histochem Cytochem 52: 179-185, 2004. Schmalbruch H and Hellhammer U. The number of nuclei in adult rat muscles with special reference to satellite cells. Anat Rec 189: 169-175, 1977. Schmalbruch H and Lewis DM. Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles. Muscle Nerve 23: 617-626, 2000. Schroeder E, Qian D, Flores C, Stewart Y, Martinez C, Terk M, and Sattler F. Body composition changes with 12 weeks of oral androgen 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. therapy in older adult men. Endocrine Society’ s 84th Annual Meeting, San Francisco, CA, 2002. 152. Schroeder E, Qian D, Stewart Y, Flores C, Martinez C, Terk M, and Sattler F. Muscle Strength and power changes with 12 weeks of oral androgen therapy in older adult men. Endocrine Society’ s 84th Annual Meeting, San Francisco, CA, 2002. 153. Schroeder ET, Singh A, Bhasin S, Storer TW, Azen C, Davidson T, Martinez C, Sinha-Hikim I, Jaque SV, Terk M, and Sattler FR. Effects of an oral androgen on muscle and metabolism in older, community-dwelling men. Am J Physiol Endocrinol Metab 284: E120-128, 2003. 154. Schroeder ET, Terk M, and Sattler FR. Androgen therapy improves muscle mass and strength but not muscle quality: results from two studies. Am J Physiol Endocrinol Metab 285: E16-E24, 2003. 155. Schroeder ET, Zheng L, Yarasheski KE, Qian D, Stewart Y, Flores C, Martinez C, Terk M, and Sattler FR. Treatment with oxandrolone and the durability of effects in older men. JAppl Physiol 96: 1055-1062, 2004. 156. Schubert W, Zimmermann K, Cramer M, and Starzinski-Powitz A. Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle. Proc Natl Acad Sci U SA 86: 307-311, 1989. 157. Schultz E, Jaryszak DL, and Valliere CR. Response of satellite cells to focal skeletal muscle injury. Muscle Nerve 8: 217-222, 1985. 158. Schultz E and McCormick KM. Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol 123: 213-257, 1994. 159. Seale P and Rudnicki MA. A new look at the origin, function, and "stem- cell" status of muscle satellite cells. Dev Biol 218: 115-124, 2000. 160. Sheehan SM, Tatsumi R, Temm-Grove CJ, and Allen RE. HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro. Muscle Nerve 23: 239-245, 2000. 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 161. Singh MA, Ding W, Manfredi TJ, Solares GS, O'Neill EF, Clements KM, Ryan ND, Kehayias JJ, Fielding RA, and Evans WJ. Insulin-like growth factor I in skeletal muscle after weight-lifting exercise in frail elders. Am J Physiol 277: E135-143,1999. 162. Sinha-Hikim I, Artaza J, Woodhouse L, Gonzalez-Cadavid N, Singh AB, Lee MI, Storer TW, Casaburi R, Shen R, and Bhasin S. Testosterone- induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. Am J Physiol Endocrinol Metab 283: El 54-164, 2002. 163. Sinha-Hikim I, Roth SM, Lee MI, and Bhasin S. Testosterone-induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. Am J Physiol Endocrinol Metab 285: E197-205, 2003. 164. Smith HK, Maxwell L, Rodgers CD, McKee NH, and Plyley MJ. Exercise-enhanced satellite cell proliferation and new myonuclear accretion in rat skeletal muscle. J Appl Physiol 90: 1407-1414,2001. 165. Snow MH. An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 186: 535-540, 1978. 166. Snow MH. The effects of aging on satellite cells in skeletal muscles of mice and rats. Cell Tissue Res 185: 399-408, 1977. 167. Snow MH. A quantitative ultrastructural analysis of satellite cells in denervated fast and slow muscles of the mouse. Anat Rec 207: 593-604, 1983. 168. Snow MH. Satellite cell distribution within the soleus muscle of the adult mouse. Anat Rec 201: 463-469, 1981. 169. Snow MH. Satellite cell response in rat soleus muscle undergoing hypertrophy due to surgical ablation of synergists. Anat Rec 227: 437-446, 1990. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 170. Tamaki T, Akatsuka A, Ando K, Nakamura Y, Matsuzawa H, Hotta T, Roy RR, and Edgerton VR. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle. J Cell Biol 157: 571-577, 2002. 171. Tamaki T, Akatsuka A, Yoshimura S, Roy RR, and Edgerton VR. New fiber formation in the interstitial spaces of rat skeletal muscle during postnatal growth. J Histochem Cytochem 50: 1097-1111, 2002. 172. Tamaki T, Uchiyama S, Uchiyama Y, Akatsuka A, Yoshimura S, Roy RR, and Edgerton VR. Limited myogenic response to a single bout of weight-lifting exercise in old rats. Am J Physiol Cell Physiol 278: Cl 143- 1152,2000. 173. Tatsumi R, Anderson JE, Nevoret CJ, Halevy O, and Allen RE. HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells. Dev Biol 194: 114-128, 1998. 174. Tatsumi R, Hattori A, Ikeuchi Y, Anderson JE, and Allen RE. Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide. Mol Biol Cell 13: 2909-2918, 2002. 175. Tatsumi R, Sheehan SM, Iwasaki H, Hattori A, and Allen RE. Mechanical stretch induces activation of skeletal muscle satellite cells in vitro. Exp Cell Res 267: 107-114,2001. 176. Tenover JS. Androgen replacement therapy to reverse and/or prevent age- associated sarcopenia in men. Baillieres Clin Endocrinol Metab 12: 419-425, 1998. 177. Thompson SH, Boxhorn LK, Kong WY, and Allen RE. Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I. Endocrinology 124: 2110-2117, 1989. 178. Tobin C and Joubert Y. The levator ani of the female rat: a suitable model for studying the effects of testosterone on the development of mammalian muscles. Biol Struct Morphog 1: 28-33, 1988. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 179. Tobin C and Joubert Y. Testosterone-induced development of the rat levator ani muscle. Dev Biol 146: 131-138, 1991. 180. Tucek S, Kostirova D, and Gutmann E. Effects of castration, testosterone and immobilization on the activities of choline acetyltransferase and cholinesterase in rat limb muscles. J Neurol Sci 27: 363-372, 1976. 181. Wanek LJ and Snow MH. Activity-induced fiber regeneration in rat soleus muscle. Anat Rec 258: 176-185,2000. 182. Watkins SC and Cullen MJ. A quantitative study of myonuclear and satellite cell nuclear size in Duchenne's muscular dystrophy, polymyositis and normal human skeletal muscle. Anat Rec 222: 6-11, 1988. 183. Welle S, Totterman S, and Thornton C. Effect of age on muscle hypertrophy induced by resistance training. J Gerontol A Biol Sci Med Sci 51: M270-275, 1996. 184. Wernig A, Bone M, Irintchev A, Schafer R, and Cullen MJ. M-cadherin is a Reliable Marker of Quiescent Satellite cells in Mouse Skeletal Muscle. Basic Appl Myol 14: 161-168, 2004. 185. Yarasheski KE. Exercise, aging, and muscle protein metabolism. J Gerontol A Biol Sci Med Sci 58: M918-922, 2003. 186. Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Increased plasma gin and Leu Ra and inappropriately low muscle protein synthesis rate in AIDS wasting. Am J Physiol 275: E577-583, 1998. 187. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins K, Thomas K, Austin T, Edwards C, Cuzzourt J, Duenzl M, Lucas PA, and Black AC, Jr. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 264: 51-62, 2001. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 188. Zammit P and Beauchamp J. The skeletal muscle satellite cell: stem cell or son of stem cell? Differentiation 68: 193-204, 2001. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX A Immunohistochemical Protocol (Rat) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunohistochemical Protocols for Satellite Cell Activation in Skeletal Muscle Cross-sections Experiment Performed b y :________________________________ Cont 0 7day 0 14day 0 28day 0 Tissue Samples: On each slide: (sections from each sample) Make sure you put in the animal # the tissue came from on the slide 1. Cut 10 pm transverse sections from muscle at -20°C and mount directly on Subbed slides. 2. Use small diameter PAP pen to draw rectangle around the sections from each animal. 3. Wash all tissue samples 3x5 min in TBS 4. Blocking reaction (1 hr min @ RT): 4% NDS in TBS-Tx1 (400pl NDS in 10ml TBS-Tx1) (NDS and TBS-Tx1 in refrigerator) 5. Incubate sections in primary antibody overnight at RT Primary Antibodies Rabbit anti-AR (N20; AN ) Santa Cruz; Rabbit anti-mMET Santa Cruz; Goat anti-M-cadherin (N-19); Santa Cruz; Incubation time Slide # 1° A b # 1 (pl) 1° Ab#2 (pl) NDS (pl) TBS- Txl (ml) Final Dilution of variable primary Antibody Overnight (RT) 1,5 Rbt AN (6.7) G tM - cadherin (2) 20 1 1.150/1:500 Overnight (RT) 2,6 Rbt AN (5) G tM - cadherin (2) 20 1 1:200/1:500 Overnight (RT) 3,7 Rbt m-Met (5) G tM - cadherin (2) 20 1 1:200/1:500 Overnight (4°C) 4,8 Rbt m-Met (10) G tM - cadherin (2) 20 1 1:100/1:500 110 (4°C; stock: 0.2 mg/ml) (4°C; stock: 0.2 mg/ml) (4°C; stock: 0.2 mg/ml) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DAY 2 1. Wash 3X5 min with TBS 2. Incubate 2 hr at RT in TBS-Tx-2 containing NDS and appropriate secondary antibody (stored at - 20°C). Slide # 2° Ab 1° Ab Host 2o Ab (Pl) NDS (pl) TBS-Tx- 2 (ml) [Secondary Ab] Dilution 1,5 Biotinylated Dnk-anti Rbt Rbt 10 20 1.000 1:200 Cy5 Dnk-anti Gt Gt 10 1:200 2,6 Biotinylated Dnk-anti Rbt Rbt 10 20 1.000 1:200 Cy5 Dnk-anti Gt Gt 10 1:200 3,7 Cy3 Dnk-anti Rbt Rbt 10 20 1.000 1:200 Cy5 Dnk-anti Gt Gt 10 1:200 4,8 Cy3 Dnk-anti Rbt Rbt 10 20 1.000 1:200 Cy5 Dnk-anti Gt Gt 10 1:200 Wash 3x5 min in TBS For slides 3&7, 4&8 skip to step 6 slide: 1&5, 2&6 Incubate 2hr at RT in TBS-Tx2 containing Streptavidin amplification of Biotin 2° Slide x-Avidin x-Avidin (pl) TBS-Tx2 (ml) [x- Avidin] pg/ml 1&5, 2&6 Cy 3-streptavidin 10 1.000 1:200 3. Wash 3x5 min in TBS 4. Place in 300nM DAPI solution (30pl lOOpM DAPI stock/ 100ml TBS) for 5 minutes 5. Wash 3x5 min in ddH2 0. 6. Coverslip with Gel Mount while the tissue is still wet. Table o f filters Slide # Cy 3 Cy 2 Cy 5 1,5 (RT) AR M-cadherin 2,6 (RT) AR M-cadherin 3,7 (RT) m-Met M-cadherin 4,8 (4°C) m-Met M-cadherin 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX B Inclusion/Exclusion Criteria Study # 2 (Human) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INCLUSION CRITERIA: a. Subjects will be recruited from the USC Health Sciences Campus. b. Be a man between the ages of 21 and 35 (younger group) or > 60 (older group) years of age. c. Be in good health (i.e., medically stable as outlined in Grieg et al., (70)). i. Subjects will have answered no to the exclusion criteria, specifically items c-w. d. Have not participated in a strength training program in the last 6 months. e. Have had no weight loss of more than 3% in the preceding two months. f. Have not previously used any anabolic agent (testosterone, anabolic steroid, testosterone precursor). g. No evidence of ischemic heart disease that would pose a health risk during the resistance exercise. Older subjects (> 60 years) will be carefully screened for evidence of ischemic heart disease. All older subjects will be required to have a stress test performed at the General Clinical Research Center (GCRC) at the USC-LAC County Medical Center prior to study entry. The stress test will be performed using standard a modified Bruce protocol developed at the University of Wisconsin and routinely used on the GCRC. The procedure utilizes real time 12-lead EKG and blood pressure monitoring. Study subjects will be coached by an exercise specialist Hans Dreyer. All stress testing for this study will be done in the GCRC Exercise Room and monitored by Dr. Fred R. Sattler, M.D (GCRC Association Program Director) who will be present in the Exercise Room during the testing procedures. A crash cart is immediately available outside of the Exercise Room. Seventeen of the 18 GCRC nurses are ACLS certified and could assist in the unlikely situation that an adverse event occurs. 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EXCLUSION CRITERIA: a. Female subjects will not be recruited due to potential protective effects of estrogen on muscle disruption. b. Subjects may not have any chronic illness (diabetes, liver diseases, kidney failure, cancer, rheumatoid arthritis). c. History of myocardial infarction, angina, or heart failure within the previous 2 years. d. Other cardiovascular illnesses: any evidence of valvular heart disease, (especially sings or symptoms of aortic stenosis or mitral regurgitation), aortic aneurysm, cardiomyopathy, uncontrolled dysrrhythmia, claudication, within the previous 10 years. e. Thrombophlebitis or pulmonary embolus within the previous 2 years. f. History of stroke. g. Any febrile illness within the previous 3 months. h. Clinically significant airflow obstruction i. Uncontrolled metabolic disease (thyroid disease). j. Major systemic disease active within the previous 2 years (e.g., cancer, rheumatoid arthritis), k. Significant emotional distress, psychotic illness or depression within the previous 2 years. 1 . Lower limb arthritis, classified by inability to perform maximal contractions of lower limbs without pain, m. Lower limb fracture sustained within the previous 2 years; upper limb fracture sustained within the previous 6 months; non-arthroscopic lower limb joint surgery within the previous 2 years, n. Any reason for loss of mobility for greater than 1 week in the previous 2 months or greater than 2 weeks in the previous 6 months, o. Resting systolic blood pressure >160 mmHg or resting diastolic blood pressure > 94 mmHg. p. Taking digoxin. q. On daily analgesia, r. Red blood cell count is low. s. Kidney function is moderately or severely impaired, t. Liver tests are significantly abnormal, u. Major surgery in the preceding 2 months, v. All subjects will be free from any known physical limitations (neuropathy, back injury or surgery, stroke, arthritis) that would prevent intense lower extremity maximum strength exercise, w. Any subjects currently on blood thinning therapy. Subjects having taken aspirin or aspirin like substances will not be allowed to undergo muscle biopsy until they have not taken any NSAIDs for at least 7 days. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX C Schedule of Events Study 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Schedule of Events. Screening to determine eligibility Stress testing for older individuals § Muscle Biopsy # 1 & Blood Draw MaximalEc centric Resistance Exercise Muscle Biopsy #2 * & Blood Draw Study day Pre-entry Pre-entry DayO Day 10-15 Day 11-16 Young X NA X X X Old X X X X X Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX D Patient Handout (Study 2) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THINGS TO REMEMBER AFTER MUSCLE BIOPSY: We will call you approximately 3-5 days after you leave the hospital to check on the healing process. You should check your biopsy site daily. If there are any problems, please call us at 323-442-2907 (Hans Dreyer, Study Coordinator office) or 323-627-7926 (Hans Dreyer Cell Phone #), or after hours or on weekends, please call the USC University Hospital Operator (323-442-8500) and ask them to page Dr. Fred Sattler, So, we can arrange to have a physician examine you. Ace Wrap: > Used to apply extra pressure to biopsy site to decrease bleeding, swelling and bruising. > Remove after 24-hour period. N O bathing/sauna/hot tub/swimming for at least 3 days. You may take a shower after 36 hours, but make sure the wound is absolutely dry afterwards. > Loosen ace wrap if it is too tight. Suture (stitch) and Steri Strip (tape to hold the wound closed): > Inspect wound daily. If there is redness (more than ~ 'A inch), drainage, swelling, or you develop fever (temperature > 100°), please call us immediately to make an appointment so that the doctor may examine the biopsy site. > Steri strips and the suture are used to close the incision site. > Remove the steri strips after 5 days, although they may fall off earlier. Things to avoid for the next 3 days: > Climbing stairs > Lifting heavy weights, especially with your legs. No cycling or long distance running. > No bathing, hot tub or swimming where incision can get wet for extended period. Things to remember: > Call/page us if signs of infection or inflammation (redness, swelling, swelling or drainage or fever occur [temperature > 100°) occur. > UseTylenol/Advil/Aleve for discomfort or excessive soreness. > Watch for blistering under the steri strips. Call us immediately if this occurs. Removal of the Suture: > When you come in for your suture removal (5-6 days after you leave the GCRC), Dr. Sattler, a Nurse or another professional will remove the suture. 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX E Delayed Onset Muscle Soreness Scale Modified Borg Scale (Study 2) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RPE Scale 6 . 7. Very, very light 8. 9. Very light 10, 11. Fairly light 12. 13. 1A Somewhat hard 15. Hard 16. 17. Very hard 18. 19. Very, very hard 20. From the above scale enter the number that corresponds to your discomfort level of Delayed Onset Muscle Soreness (DOMS) into the appropriate column. Note 0 Hours = right after the maximal eccentric exercise 0 Hours 6 Hours 24 Hours 48 Hours 72 Hours Hand in to Hans when finished. Thanks Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX F Immunohistochemical Protocol General Procedures (Study 2) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunohistochemical Protocols for Satellite Cell Activation in Skeletal Muscle Cross sections of Young (21-35) and Older (60+) year old men Date Young & Old Experiment # 21 CD56 & Laminin Experiment Performed by:_____ Hans_______________________ 4858 Bx 1&2 2040 Bx 2 Tissue Samples: On each slide: (sections trom eacn sample) Hour 1 6. Cut 10 pm transverse sections from muscle at -20°C and mount directly on Subbed slides. 7. Use small diameter PAP pen to draw rectangle around the sections from each animal. 8. Wash all tissue samples 3x5 min in TBS 9. Blocking reaction (1 hr min @ RT): 4% NDS in TBS-Tx1 (400pl NDS in 10ml TBS-Tx1) (NDS and TBS-Tx1 in refrigerator) Primary Antibodies Mouse anti-CD56 (NCAM) DAKOCytomation;(4°C; stock: 8.2 mg/ml) Rabbit anti-laminin DAKOCytomation;(4°C; stock: 4.1 mg/ml) Incubation time Slide # 1° Ab # 1 m 1° Ab # 2 m NDS m TBS- Txl (ml) Final Dilution of variable primary Antibody 2 hours (RT) 1 Mouse CD56 (20) Rbt Laminin (1) 40 2 1:100/1:2000 2 hours (RT) 2 Mouse CD56 (20) Rbt Laminin (1) 40 2 1:100/1:2000 2 hours (RT) 3 Blank Blank 40 2 1:2000/1:2000 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hour 4-6.5 7. Wash 3X5 min with TBS 8. Incubate 2 hr at RT in TBS-Tx-2 containing NDS and appropriate secondary antibody (stored at -20°C). Slide # 2° Ab 2° Ab m NDS (til} TBS-Tx-2 (ml) TSecondarv Abl Dilution 1:200 1 u 2 Cy3 Dnk-anti Mouse 40 80 4.0 u 3 Cy2 Dnk-anti Rbt « tt u 9. Wash 3x5 min in TBS 10. Place in 300nM DAPI solution (30pl 100pM DAPI stock/ 100ml TBS) for 5 minutes 11. Wash 3x5 min in ddH20. 12. Cover-slip with Gel Mount while the tissue is still wet. Table of filters Slide # Cy 3 Cy 2 Cy 5 1 & 2 CD-56 (NCAM) Laminin 3 Cy3 secondary Cy2 secondary NOTES 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX G Immunohistochemical Protocol Fiber Typing (Study’s 1 & 2) 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunohistochemical Protocols for Satellite Cell Activation in Skeletal Muscle Cross-sections of Young (21-35) and Old (60+) year old men D ate______________ Experiment # 2 2 Fiber Typing Experiment Performed by:________________________ Tissue Samples: On each slide: (sections from each sample) Make sure you put in the animal # the tissue came from on the slide 10. Cut 10 pm transverse sections from muscle at -20°C and mount directly on Subbed slides. 11. Use small diameter PAP pen to draw rectangle around the sections from each animal. 12. Wash all tissue samples 3x5 min in TBS 13. Blocking reaction (1 hr min @ RT): 4% NDS in TBS-Tx1 (400pl NDS in 10ml TBS-Tx1) (NDS and TBS-Tx1 in refrigerator) 14. Incubate sections in primary antibody overnight at RT Primary Antibodies Mouse anti MyHCi Sigma; (-20°C; stock: recon) Rabbit anti-laminin DAKO; (4°C; stock: 4.2 mg/ml G O * ■ § S 4fc u 1° A b# 1 1° A b#2 NDS TBS- Final Dilution of -o § a t-H IS 55 (H i) (Hi) ( p l) Txl (ml) variable primary Antibody Overnight (RT) All Ms MyHC-I (1) Rbt Laminin 0 ) 40 2 1:2000/1:2000 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DAY 2 (Slides 1-5 only) 13. Wash 3X5 min with TBS 14. Incubate 2 hr at RT in TBS-Tx-2 containing NDS and appropriate secondary antibody (stored at -20°C). Slide # 2° Ab 1° Ab Host 2° Ab 0 0 ) NDS 00} TBS-Tx- 2 (ml) rSecondarv Ab] Dilution All Cy3 Dnk-anti Ms Ms 10 20 1.000 1:200 Cy2 Dnk-anti Rbt Rbt 10 1:200 15. Wash 3x5 min in TBS 16. Place in 300nM DAPI solution (30pl 100pM DAPI stock/ 100ml TBS) for 5 minutes 17. Wash 3x5 min in ddH20. 18. Coverslip with Gel Mount while the tissue is still wet. Table of filters Slide # Cy 3 Cy 2 Cy 5 All MyHC-, Laminin 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX H Power Estimates for Future Studies on Satellite Cells in Humans Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Using the nQuery Advisor statement™ v. 1.0 software package the following power estimates were obtained from the results of study 2 (Chapter 4) of this document. Sample Population of Interest: Young Men Variable of Interest: Satellite Cells/Muscle Fiber A sample size of four (4) in each group will have 80% power to detect a difference in means of -0.103 (the difference between Pre-Exercise biopsy, pi, of 0.073 and Post- Exercise biopsy, p2, of 0.176) assuming that the common standard deviation is 0.041 using a two group /-test with a 0.050 two-sided significance level. Sample Population of Interest: Young Men Variable of Interest: Satellite Cells Proportions [SC/(SC + Myonuclei)] x 100 A sample size of five (5) in each group will have 80% power to detect a difference in means of -3.190 (the difference between Pre-Exercise biopsy, pi, of 2.860 and Post- Exercise biopsy, p2, of 6.050) assuming that the common standard deviation is 1.340 using a two group /-test with a 0.050 two-sided significance level. Sample Population of Interest: Older Men Variable of Interest: Satellite Cells/Muscle Fiber A sample size of nine (9) in each group will have 80% power to detect a difference in means of -0.030 (the difference between Pre-Exercise biopsy, pi, of 0.067 and Post-Exercise biopsy, p2, of 0.097) assuming that the common standard deviation is 0.021 using a two group t-test with a 0.050 two-sided significance level. Sample Population of Interest: Older Men Variable of Interest: Satellite Cells Proportions [SC/(SC + Myonuclei)] x 100 A sample size of five (5) in each group will have 80% power to detect a difference in means o f-1.310 (the difference between Pre-Exercise biopsy, pi, of 2.860 and Post- Exercise biopsy, p2, of 4.120) assuming that the common standard deviation is 0.593 using a two group t-test with a 0.050 two-sided significance level. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NOTE TO USERS This reproduction is the best copy available. ® UMI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3155401 Copyright 2004 by Dreyer, Hans Christian All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3155401 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The influence of body weight and composition, pubertal status, tobacco use and exposure, physical activity and muscle strength on bone mass of Chinese adolescents

PDF

Potential factors contributing to diaphragm myopathy in congestive heart failure

PDF

Factors contributing to patellofemoral joint stress: a comparison of persons with and without patellofemoral pain

PDF

The influence of eccentric resistance training on bone mass and biochemical markers in young women

PDF

Motor skill learning in individuals with Parkinson's disease: Consideration of cognitive and motor demands

PDF

Investigating the metabolic and cellular parameters of lipid metabolism in skeletal muscle: Effects of dietary, pharmacological and exercise challenges

PDF

The role of the sensorimotor cortical system in skill acquisition and motor learning: a behavioral study

PDF

Biomechanical and neuromuscular aspects of non-contact ACL injuries: The influence of gender, experience and training

PDF

At what age are gait characteristics mature? Evaluation of gait kinematics, kinetics, and intersegmental dynamics in 7 year-old children

PDF

Cardiovascular fitness, exercise, and metabolic disease risk in overweight Latino youth

PDF

The role of the vasti in patellar kinematics and patellofemoral pain

PDF

Functional brain correlates for premovement planning and compensatory adjustments in rapid aimed movement

PDF

The influence of patella alta on knee extensor mechanics and patellofemoral joint stress

PDF

The interaction between explicit knowledge and implicit motor -sequence learning following focal brain damage

PDF

HER-2/neu-mediated cell migration and invasion

PDF

Quantifying musculoskeletal load and adaptation: Biomechanical consideration

PDF

Characterization of neural mechanisms involved in hypoglycemic detection at the portal vein

PDF

The physiological effects of a single bout of eccentric versus concentric resistance exercise

PDF

Elucidation of the origin and contribution of hepatoportal and central nervous system glucose sensors mediating hypoglycemic detection

PDF

Virus impacts upon marine bacterial and diazotroph assemblage composition

Asset Metadata

Creator Dreyer, Hans Christian (author)

Core Title Satellite cell proliferation: two models and two anabolic stimuli

School Graduate School

Degree Doctor of Philosophy

Degree Program Biokinesiology

Degree Conferral Date 2004-12

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, animal physiology,biology, cell,health sciences, rehabilitation and therapy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-567992

Unique identifier UC11340200

Identifier 3155401.pdf (filename),usctheses-c16-567992 (legacy record id)

Legacy Identifier 3155401.pdf

Dmrecord 567992

Document Type Dissertation

Rights Dreyer, Hans Christian

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA