Charlotte A. Peterson - US grants

Molecular mechanisms that lead to the progressive loss of muscle size and strength with age in humans have not been elucidated. Age-related changes in the ability of adult myoblasts to proliferate and differentiate in response to muscle damage could reduce muscle size. Beta-enolase, a muscle-specific glycolytic enzyme, is expressed in proliferating myoblasts of adult, but not fetal muscle. This is in contrast to most muscle- specific genes which are expressed upon differentiation, under the control of the MyoD family of regulators. The goal of this research is to identify the molecular mechanisms that control beta-enolase gene expression in adult myoblasts and determine if changes in gene regulation occur with age. This will be accomplished by defining the cis-regulatory DNA sequences responsible for controlling the beta-enolase gene. Expression of reporter gene constructs containing different amounts of DNA flanking the beta- enolase gene will be assayed following transfection into muscle and nonmuscle cells. Proteins that bind to the regulatory DNA sequences will be isolated from an adult human myoblast cDNA expression library. Beta- enolase is also expressed in differentiated myofibers and accumulation of beta-enolase mRNA changes with age in muscle tissue. This may be indicative of more global changes in the metabolic activity of the muscle since several lines of evidence suggest that genes encoding glycolytic enzymes may be coordinately regulated. The molecular mechanisms that control beta-enolase gene expression in myofibers will also be explored. The role of the MyoD family of proteins in regulating beta-enolase expression following differentiation will be ascertained. Finally, the human homology to the yeast GCR1 gene product, which has been shown to be necessary for the coordinate regulation of yeast glycolytic enzyme genes, will be isolated. A GCR1 DNA probe, generated using oligonucleotide primers to published sequence and PCR, will be used to screen a human myoblast cDNA library under low stringency. Therefore, in addition to identifying factors involved in muscle-specific expression, factors involved in the coordinate regulation of metabolic enzyme gene expression will be identified. Changes in the activity of these factors may contribute to the loss of muscle function with age.

The project described here is a step towards my long term career goal which is to contribute fundamental knowledge regarding the molecular mechanisms underlying changes in gene expression in muscle as a result of injury or trauma and how this process is affected by aging. We have focused our studies on defining the regulatory mechanisms that control gene expression in satellite cells, the adult myoblasts that proliferate and differentiate in response to muscle injury, thereby regenerating damaged fibers. We are currently mapping the cis-regulatory elements controlling the muscle-specific enolase gene that, unlike most muscle- specific genes, is expressed at high levels in satellite cells. We propose here to isolate trans-acting factors that bind to the cis-regulatory DNA sequences by screening a bacterial expression library. We also propose to establish yeast as an experimental system and utilize genetic selection in yeast to isolate beta-enolases regulators, and proteins that interact with them to modify their activity. This award will guarantee the time required for me to gain experience and proficiency in yeast genetics.

DESCRIPTION (provided by applicant): This project will explore a potentially novel mechanism contributing to muscle atrophy with age. Our data show that the myogenic progenitors present in muscle that are primarily responsible for postnatal muscle growth, repair and maintenance are activating an adipocyte gene program with age. This observation is particularly intriguing in that it suggests that common mechanisms may control loss of muscle mass and bone density with age, as it has been shown that adipocytes increase in the bone marrow stroma with age at the expense of osteoblast progenitors. We hypothesize that muscle atrophy and the increased fat content in muscle with age may be due, at least in part, to changes in the potential of myogenic progenitors. Aim 1 will explore myogenic progenitor potential in vivo to determine if the change in differentiation potential of the cells impacts muscle phenotype and if cell fate is affected by the muscle environment. Aim 2 will focus on the Wnt signaling pathway that negatively regulates adipocyte gene expression. Gene transfer will be used to inhibit or activate the Wnt pathway in myogenic progenitors to elucidate its role in muscle aging. In Aim 3, we will determine if the changes in myogenic progenitor potential with age observed in mice also occurs in humans. Progenitors will be isolated from muscle of aged and young volunteers who have been physiologically and functionally characterized, and their differentiation potential assessed. Finally, in Aim 4, gene expression in myogenic progenitors and bone marrow stromal cells from both humans and mice will be examined using microarray technology to identify patterns of expression characteristic of mesenchymal progenitor cells in adult tissues. We will identify changes in gene expression that are indicative of a change in potential with age. In summary, changes in mesenchymal progenitor cell fate will be characterized and their contribution to the decline in the musculoskeletal system established. Putative regulators of the change in fate will be identified and future studies will evaluate their potential as targets for therapeutic intervention to prevent, slow, or even reverse the loss in the musculoskeletal system with age.

DESCRIPTION (provided by applicant): The goal of this proposal is to perform a comprehensive analysis of a novel form of resistance exercise training as a countermeasure to offset the loss in musculoskeletal structure and function after exposure to microgravity. Rats, continuously maintained in a hindlimb suspended state, will be subjected to resistance training using flywheel technology. We hypothesize that this form of resistance training, that is gravity-independent, will prevent muscle atrophy and loss of bone mineral density normally associated with hindlimb suspension. Force generated during training will be quantitated using a load cell and correlated with skeletal muscle mass, fiber cross-sectional area, and performance in Aim I. To identify specific mechanisms responsible for the changes in muscle properties in response to flywheel training, the dynamics of muscle protein homeostasis will be systematically quantitated. Aim 2 will explore the effects of flywheel training on bone mass, density and strength. Quantitation of the strain required for maintenance of bone properties using a strain gage will be correlated to muscle force to allow the development of a training regimen that is beneficial to the entire musculoskeletal system. The relative contribution of apoptotic muscle fiber nuclear loss and osteocyte apoptosis to loss of musculoskeletal function during hindlimb suspension and the effect of flywheel training will be determined in Aim 3. Finally, in Aim 4, muscle and bone marrow stem cells will be isolated and proliferation and differentiation analyzed to determine if changes in these cellular processes potentially contribute to the response to hindlimb suspension and flywheel resistance training. This approach will provide a basic understanding of mechanisms contributing to maintenance of the musculoskeletal system via resistance exercise during microgravity. Our experiments are directly relevant to studies being carried out in humans to determine efficacy of flywheel training protocols. Together, results may provide insight into the use of resistance exercise as a countermeasure to the loss of muscle and bone that occurs during space flight.

DESCRIPTION (provided by applicant): Resident macrophages in adipose tissue account for much of the cytokine expression with obesity. We described the expression of thrombospondin (TSP1), a pleiotropic adipokine, in insulin resistant subjects. Through its anti-angiogenic properties and its ability to activate TGF2, TSP1 may be an important part of the adipocyte/macrophage interaction that results in simultaneous impairment of angiogenesis and increased TGF- 2-mediated fibrosis and inflammation. We recently demonstrated that macrophages also infiltrate skeletal muscle with obesity that is strongly correlated with insulin resistance. Furthermore, the presence of macrophages in co-culture with muscle cells, in the presence of palmitic acid to mimic an obese environment, results in a synergistic increase in the expression and secretion of inflammatory cytokines from muscle cells, all of which impair insulin action. We hypothesize that macrophages in both muscle and adipose tissues promote the development of metabolic syndrome during obesity. To test this hypothesis, adipose (Aim 1) and muscle tissue (Aim 2) from obese, insulin resistant compared to normal human subjects will be characterized. Macrophage number, the proportion of macrophages that are classically versus alternatively activated, and the proportion of macrophages in crown-like structures (in adipose) will be quantified, along with markers of the TGF2 pathway, fibrosis, hypoxia, tissue remodeling and vascularity. To specifically determine the role of TSP1 and the TGF2 pathway in insulin resistance, adipose and muscle tissue fibrosis, hypoxia, and macrophage function will be analyzed in high fat fed TSP1 null mice in Aim 3. A peptide that specifically blocks TSP1- mediated TGF2 activation will also be used in wild type, high fat fed mice to test the hypothesis that macrophage infiltration and fibrosis in adipose and muscle will be ameliorated in the absence of TSP1, resulting in improved insulin sensitivity. Aim 4 is the intervention aim in which the mechanisms underlying the beneficial effects of aerobic exercise training on insulin sensitivity will be determined. Following 12 weeks of training in obese and lean subjects, changes in muscle and adipose macrophage number, activation state, the TSP1/TGF2 pathway, and inflammation in response to a single bout of eccentric exercise will be quantified relative to insulin sensitivity. Finally, in Aim 5, the effects of different macrophage populations on adipocytes and muscle cells will be studied in vitro. The expression of genes involved in adipogenesis, extracellular matrix, and the TGF2 pathway will be monitored in adipocytes following macrophage co-culture. These will also be quantified in muscle cells, as well as the response to insulin. We will determine whether mechanical stimulation of myotubes to mimic exercise will alter the muscle cell response to macrophages. PUBLIC HEALTH RELEVANCE: The American population is experiencing an explosion in obesity, metabolic syndrome and diabetes, and this national trend is magnified in Kentucky and the Southeast. The cost of caring for diabetes and its complications is enormous. Impaired glucose tolerance and metabolic syndrome are preludes to the development of type 2 diabetes, represent a risk factor for coronary artery disease, and are more prevalent than diabetes itself. These studies are intended to examine fundamental mechanisms of insulin resistance and inflammation, and involve studies in humans, mice, and cell culture. New insight into these fundamental mechanisms may form the basis for new treatments.

DESCRIPTION (provided by applicant): Fibromyalgia (FM) is a clinical state of widespread musculoskeletal pain with multiple tender points that is most common in postmenopausal women. Fatigue, particularly post-exertional, is an additional hallmark of the syndrome. FM likely has no single etiology and may have neuroendocrine, neurologic, immune and musculoskeletal components. Fatigue and fatigability also contribute directly to functional dependence and activity limitations that impact quality of life among the aged. Although associated with co-morbidities, fatigue in the elderly is often not associated with an identified medical cause. The relationship among fatigue associated with FM, fatigue associated with disease, and exacerbation of fatigue symptoms with age are unexplored. The goal of this pilot study is to identify defects in muscle physiology of older FM patients, as well as older healthy but fatigable individuals, which may contribute to this symptom. Older normal healthy women and women who are healthy but prove most fatiguable during testing, as well as older women diagnosed with FM, will be studied. Aim 1 will test the hypothesis that decreased blood flow and reduced muscle oxygenation contribute directly to pain and post exercise fatigue. Using novel, noninvasive near-infrared diffuse optical spectroscopies, muscle blood flow and oxygen saturation will be quantified before, during and after an acute bout of exercise. To identify mechanisms underlying reduced tissue oxygenation, muscle microvasculature will be analyzed in Aim 2. Vascular density and endothelial function will be assessed in muscle biopsies by immunofluorescent detection of endothelial cell antigens and detection of endothelial alkaline phosphatase, and non-invasively with the flow-mediated dilatation (FMD) test and optical probe. Aim 3 will test the hypothesis that the long term consequence of reduced muscle oxygenation may be compromised mitochondrial function. Assays of mitochondrial oxidative phosphorylation from muscle biopsies will be performed, including respiration rates and complex I-IV activities. Aberrant accumulation of mitochondrial metabolites may alter muscle vasodilatory properties, further reducing oxygen availability contributing to the pain and fatigue of FM, as well as fatigue in the elderly. PUBLIC HEALTH RELEVANCE: Fibromyalgia is increasingly common in postmenopausal women. As FM is commonly associated with pain, fatigue, sleep disturbances, depression, psychological and mood disorders, and cognitive deficiencies, individuals with FM may suffer from temporary to permanent debilitation. This project will explore the role of altered muscle physiology as a contributor to the pain and fatigue experienced by fibromyalgic patients, as well as older healthy women experiencing fatigue.

DESCRIPTION (provided by applicant): The goal of this proposal is to develop and characterize a novel mouse line which will allow for the conditional, genetic labeling of satellite cells in adult skeletal muscle. Satellite cells are the myogenic stem cell of adult skeletal muscle and have been most studied during post-natal growth and during muscle regeneration following injury. Beyond speculation, the role of satellite cells in the maintenance of skeletal muscle throughout life has yet to be directly studied due to the difficulty of accurately identifying satellite cells and the inability to stably track their frequency and behavior over time. To overcome this obstacle, in Aim 1 we will generate the Pax7-GNZ mouse by crossing the conditional, satellite cell-specific driver/inducer mouse line (Pax7-CreER) with a nuclear-localized GFP-lacZ reporter mouse line (Rosa26-GNZ). A major strength of the proposal is that the parental strains required to generate the Pax7-GNZ line already exist. Characterization of the Pax7-GNZ mouse line will entail quantifying the specificity and magnitude to which the reporter gene effectively marks satellite cells. The role of satellite cells in maintaining muscle mass will then be determined by inducing reporter gene expression in 3 month old mice which will be analyzed at later time points throughout the lifespan of the mice. Aim 2 will begin to determine the contribution of satellite cells to the compromised ability of aging muscle to adapt to changes in demand. The Pax7-GNZ mice will be used to quantify satellite cell dynamics with muscle hypertrophy induced by synergist ablation and re-growth following muscle atrophy induced by hindlimb unloading. The Pax7-GNZ mouse line represents a powerful, unique genetic tool that will allow for the first time, a comprehensive and accurate quantification of satellite cell dynamics in the maintenance of muscle during normal muscle aging, as well as during periods of altered demand such as hypertrophy and restoration of mass following atrophy. Furthermore, results obtained will serve as the foundation for future studies using a genetic mouse model to examine the impact on muscle plasticity of specifically ablating satellite cells in adult skeletal muscle. These studies will define the function of satellite cells in skeletal muscle plasticity and homeostasis and how it may change with age. PUBLIC HEALTH RELEVANCE: Almost one-third of the elderly suffer from the debilitating condition of frailty. The cause of geriatric frailty is not known but a contributing factor is sarcopenia, the progressive loss of muscle mass with age. Defects in satellite cells, the primary stem cell of adult skeletal muscle, are thought to play a central role. The goal of this proposal is to develop and characterize a novel mouse model to track satellite cells in aging skeletal muscle with the aim to better understand their role in sarcopenia. The long-term goal of the research project is to develop a therapeutic intervention to prevent sarcopenia and reduce the incidence of frailty in the elderly.

DESCRIPTION (provided by applicant): The loss of skeletal muscle mass is of clinical importance because it is associated with increased morbidity and mortality, as well as a marked deterioration in the quality of life. A broad patient population is affected by significant losses in muscle mass including those afflicted by various systemic diseases (cancer, sepsis, HIV- AIDS), chronic physical inactivity as a result of long term bed rest, rheumatoid arthritis and limb immobilization, and sarcopenia, the age associated loss in muscle mass and strength. Satellite cells are currently an attractive therapeutic target given their stem cell characteristics and essential role in post-natal muscle growth and regeneration. What remains controversial is the necessity of satellite cells in other aspects of muscle plasticity such as hypertrophy, re-growth following atrophy and muscle maintenance with aging. In an effort to resolve this fundamental issue, a novel mouse line was created which enables the specific ablation of satellite cells in mature skeletal muscle. The Pax7-DTA mouse will be used to investigate the physiological function of satellite cells in skeletal muscle hypertrophy (Aim 1) and re-growth following muscle atrophy (Aim 2). The results from the proposed experiments are expected to provide fundamental knowledge on the function of satellite cells in adult skeletal muscle plasticity which will help define the therapeutic value of satellite cells in treating the loss of muscle mass in various clinical conditions. PUBLIC HEALTH RELEVANCE: Loss of muscle, a common symptom associated with many chronic diseases, physical inactivity or aging, negatively impacts a person's quality of life, increases the susceptibility to other complications of disease and can even lead to death. Treatments designed to restore or prevent muscle loss have in part focused on using muscle stem cells referred to as satellite cells. The goal of the proposed research is to determine if satellite cells are necessary for adult muscle growth and thus, are an appropriate target for therapy for skeletal muscle loss.

DESCRIPTION (provided by applicant): This proposal requests financial support from the National Institute on Aging for the 2014-2015 Annual Meetings organized by the Biological Sciences (BS) Section of the GSA. Our guiding principle to organizing the 2014-2015 meetings is that the highest quality and most relevant science in the aging field will emerge when researchers understand the issues of human aging from geriatricians, psychologists and sociologists. In turn, improvements in clinical care and the health of nation will be facilitated when geriatricians, psychologists and sociologists understand the basic mechanisms of aging and the efficacy of interventions designed to ameliorate aging and age-related disease. Furthermore, we are cognizant of our responsibility to provide BS members, as well as other members of the GSA, the latest cutting-edge, high quality science that ultimately will improve the research of all who attend. The annual meeting of GSA is the only meeting in the country that has the ability to foster such an interdisciplinary approach to aging research. The GSA BS Section has re-dedicated itself to organizing the highest quality scientific program at the annual meeting, thereby providing a forum to engender interaction and exchange of ideas among scientists from disparate fields. The goal is to raise awareness of recent, fundamental advances in our understanding of aging biology and facilitate translation into clinical interventions. We propose a single-track program featuring emerging concepts in the basic biology of aging with the highest translational potential that will promote discussion and networking among speakers and conference attendees across sections, particularly between the BS and Health Sciences Sections. The meeting will also provide opportunities for talented junior investigators to have a prominent role in BS symposia. Scientific sessions and overall meeting logistics will be evaluated to implement quality improvement for the subsequent year and to provide continuity for symposium themes.

DESCRIPTION (provided by applicant): The loss of skeletal muscle mass and strength with advancing age reduces quality of life and is a major factor limiting an elderly person's chance of living independently. Progressive resistance exercise training (PRT) is the most effective intervention identified to increase muscular strength and combat muscle atrophy of aging; however, overall the muscle response to PRT is blunted and highly variable in the elderly. Our research team has determined that the abundance of anti-inflammatory, alternatively activated M2 macrophages in muscle predicts response to PRT in the elderly; those with the highest number of M2 macrophages and lowest inflammatory gene expression prior to the start of training gained the most mass. Further, reexamination of muscle biopsies obtained in a study on insulin resistance showed that metformin treatment increased M2 macrophage abundance, and decreased inflammatory cytokine gene expression. These provocative findings have led us to our central hypothesis that adjuvant metformin may improve the responses to PRT in the elderly by altering the muscle tissue inflammatory environment, thereby enhancing mechanisms that drive PRT-induced myofiber hypertrophy. In Aim 1, we will determine if metformin treatment augments skeletal muscle size and strength gains in conjunction with PRT in older, functionally limited adults. Participants will be recruited and randomized to receive either placebo or metformin for 2 weeks followed by a 14 week PRT program with continued drug/placebo treatment. Gains in muscle size and strength will be quantified. In Aim 2, we will identify cellula and molecular responses in muscle to metformin which are associated with improved response to PRT. Muscle macrophages, inflammatory gene expression and anabolic and inflammatory signaling pathways will be examined in muscle biopsies. Finally, mechanisms underlying metformin effects on muscle response to training, using a human muscle cell culture system modeling exercise and the muscle microenvironment, will be explored in Aim 3. Prospective identification of individuals likely to be refractory to routine exercise programs, and determining the effectiveness of metformin in improving muscle growth response to PRT, may contribute to the development of an affordable, personalized approach to maintain or restore skeletal muscle mass and strength, thereby promoting longer healthspan.

? DESCRIPTION (provided by applicant): Under the current award, we have acquired exciting new evidence that satellite cells are necessary for the proper remodeling of the extracellular matrix during hypertrophy. We reported that in muscle depleted of satellite cells there was a significant increase in fibrosis that was associated with a blunted long-term hypertrophic response. Our preliminary data show that activated satellite cells/myogenic progenitor cells (MPCs) are capable of repressing the synthesis of extracellular matrix components by fibroblasts through exosomal delivery of microRNAs. We hypothesize that the loss of satellite cells removes this brake leading to the over-production of collagen and fibrosis, ultimately limiting long-term hypertrophic growth. We further hypothesize that fibrosis as a result of loss of satellite cells preferentially limits growth in fast muscle fibers, whereas growth of slow fibers i limited by myonuclear domain size. Thus, fast fibers display a relatively flexible myonuclear domain, whereas slow fibers may have a more stringent requirement for satellite cells for growth. The objectives of this proposal are to use genetic mouse models and in vitro cell culture to investigate these novel roles for satellite cells in skeletal muscle adaptation by pursuing the following aims. Aim 1 will determine the mechanisms underlying satellite cell regulation of extracellular matrix remodeling during skeletal muscle hypertrophy. Aim 2 will determine if fibrosis attenuates long-term hypertrophy in satellite cell-depleted muscle, and the influence of fiber type composition. Aim 3 will determine if there is a fiber type-specific requirement for satellite cell fusion during skeletal muscle hypertrophy. Our published work and new preliminary data clearly show that our understanding of satellite cell function in adult skeletal muscle adaptation remains incomplete. The studies described in this proposal address this fundamental gap in our knowledge and are expected to provide critical information necessary to more effectively evaluate the use of satellite cells as a therapeutic agent to prevent or restore the los of skeletal muscle mass associated with dystrophies, cancer, age and rehabilitation following disuse.

Abstract A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength. Although the etiology of sarcopenia remains to be determined, studies in humans and rodents have reported a strong correlation between the loss and/or dysfunction of satellite cells and sarcopenia. Despite the correlation between declining satellite cell-dependent regenerative capacity and age, no studies to date have directly tested this relationship to determine if the loss of satellite cells causes sarcopenia. To test this idea, we depleted (>85%) satellite cells in five month old mice to a level dramatically lower than that observed with normal aging. A detailed analysis of multiple muscles through 24 months of age revealed that, despite significantly reduced regenerative capacity, the life-long depletion of satellite cells did not accelerate nor exacerbate sarcopenia; however, the depletion of satellite cells at a young age was associated with a significant increase in fibrosis in old mice. These highly provocative findings, together with our data on the fiber-type specific role of satellite cells in response to exercise, reveal our limited understanding of how aging affects the function of satellite cells in skeletal muscle maintenance, the development of fibrosis and in response to a growth stimulus; addressing these fundamental gaps in our knowledge clearly requires new tools. Towards this end, we will utilize a novel mouse strain (Pax7-H2B-GFP) that will allow us to track satellite cell dynamics for the first time in adult skeletal muscle aging. To better understand how aging and exercise affects satellite cell dynamics and the regulation of fibrosis, the following aims will be pursued: 1) determine how age and life-long exercise affects satellite cell dynamics in the maintenance of skeletal muscle, 2) determine how age and life-long exercise affects satellite cell regulation of fibrosis and 3) determine how age affects satellite cell dynamics in response to a growth stimulus. The approaches described herein use powerful, new genetic tools to determine how aging and life-long exercise alters the function of satellite cells in skeletal muscle homeostasis, regulation of fibrosis and adaptability. The development of the Pax7-H2B-GFP mouse represents a long sought-after method for tracking satellite cells, especially following fusion into the myofiber. This novel mouse strain will allow us to address formally intractable questions regarding how satellite cell dynamics are affected by age and life-long exercise. Such fundamental knowledge is necessary to critically evaluate the therapeutic value of satellite cells for the treatment of muscle mass loss and function associated with aging.

Summary Exosomes are small extracellular vesicles that have emerged over the past few years as novel mediators of intercellular communication. We recently reported that exosomes released from activated skeletal muscle stem cells regulate extracellular matrix remodeling during muscle hypertrophy through the transport of skeletal muscle-specific miR-206 to fibroblasts. The discovery of this novel role for muscle stem cells inspired us to further explore if exosomes are released from muscle cells in response to resistance exercise and, if so, their impact on target tissues. Based on preliminary data, we hypothesize that enhanced adrenergic signaling in adipose tissue in response to resistance exercise is mediated in part by exosomal muscle-specific miR-1 activation of ?3-adrenergic receptor (?3-AR) expression. In particular, we have developed a working model in which exercise causes skeletal muscle cells to release exosomes containing muscle-specific miR-1 that transport miR-1 to adipocytes. As a result, CAATT/enhancer binding protein alpha (C/EBP?) activates ?3-AR gene expression through miR-1-mediated repression of AP-2?, thereby enhancing catecholamine sensitivity of adipocytes and promoting the release of fatty acids and glycerol into the circulation as the result of increased lipolysis. The purpose of the proposed studies is to test our working model by pursuing the following aims in both mice and humans: Aim 1: Determine if exercise-induced exosomal miR-1 enhances adipocyte adrenergic signaling through activation of ?3-AR expression; Aim 2: Determine if human skeletal muscle fiber-derived exosomes directly promote adipocyte lipolysis through enhanced ?-adrenergic signaling; Aim 3: Determine if an acute bout of resistance exercise in humans promotes miR-1-mediated adipocyte lipolysis. Our preliminary data provide the first evidence demonstrating that resistance exercise-induced exosomes mediate the beneficial effect of exercise on adipose tissue metabolism. A better understanding of the role of exosomes in the systemic adaptations that occur in response to resistance exercise are expected to provide the fundamental knowledge necessary to use exosomes as a platform for the delivery of exercise mimetics to treat obesity.

Lower extremity peripheral artery disease (PAD) significantly affects aging populations and results in functional impairment. Although the clinical importance of finding efficacious interventions for PAD is well-recognized, few medical therapies are currently available. PAD is diagnosed using the ankle brachial index (ABI), a measure of blood flow to the lower extremities. Lower ABI is associated with worse function; however, low ABI alone cannot fully explain functional impairments in PAD. Small studies have reported oxidative stress, mitochondrial dysfunction and/or fiber damage in gastrocnemius muscle biopsies from PAD patients, suggesting skeletal muscle perturbations may contribute to functional decline. We reported highly variable fiber type composition and fiber type grouping in a small cohort of PAD patients, and observed lack of intermyofibrillar mitochondria (IMFM-) in oxidative, myosin heavy chain (MyHC) type I fibers. We have provocative new preliminary data suggesting variability in response to ongoing denervation, and in fiber type and mitochondrial adaptations, with PAD. The purpose of this study is to define specific characteristics of muscle in PAD associated with impaired walking performance through detailed immunohistochemical analyses of 400 baseline gastrocnemius muscle biopsies stored in the Northwestern biorepository, collected from 9 different clinical trials. This biorepository of muscle from PAD patients is one-of-a-kind and is associated with detailed clinical and functional characteristics of the donors. We hypothesize that variability in fiber size, fiber type and mitochondrial adaptations in response to ischemia-reperfusion damage and denervation in individuals with PAD will have value in predicting walking impairment. In Aim 1, we will quantify the proportion of IMFM- areas in type I fibers with normal type I MyHC abundance, or accumulation of type IIX MyHC and/or LC3, a marker of autophagy, and determine associations with fiber type composition and fiber size, as well as relationships of muscle features to walking performance in PAD. We hypothesize that LC3 will co-localize with IIX MyHC in IMFM- areas, suggesting both incomplete autophagic clearance of IIX MyHC and mitochondrial biogenesis during fiber transition from type IIX to type I as a result of denervation and reinnervation. In Aim 2 we will quantify denervated, NCAM+ fibers and fibers with elevated oxidative damage markers by fiber type. We hypothesize that denervation in PAD will preferentially affect fibers expressing IIX MyHC and that only IMFM- areas that accumulate IIX MyHC will be NCAM+. In Aim 3 we will perform predictive modeling of PAD disease severity and functional impairment using morphological characteristics of muscle quantified in Aims 1 and 2 as biomarkers in conjunction with supervised classification approaches. In Aim 4 we will test the hypothesis that baseline muscle characteristics will predict longitudinal functional outcomes at 6-month follow up. This model will provide a powerful tool to aide in identifying biologic processes for targeted interventions and to assess the mechanism of action and effectiveness of current pharmacological and exercise interventions in ongoing PAD clinical trials.

Project Summary The loss of skeletal muscle mass with age is of clinical importance because it is associated with increased morbidity and mortality, as well as a marked deterioration in the quality of life. There is also a heightened interest in the identification of cellular and molecular mechanisms responsible for the lack of an anabolic response of aged muscle to hypertrophic stimuli. The use of satellite cells to treat loss of skeletal muscle mass is considered a promising therapeutic strategy given their stem cell characteristics and essential role in post- natal muscle growth and regeneration. The results of our studies have prompted us to perform mechanistic analyses of both of the well-known function of satellite cells; fusion to myofibers to provide additional nuclei for hypertrophic growth, to other functional consequences of satellite cell expansion that occurs in response to various exogenous stimuli such as exercise, as the increase in satellite cell abundance in response to mechanical overload far exceeds myonuclear accretion associated with increased myofiber size. We reported satellite cells were necessary for optimal long-term hypertrophic growth of skeletal muscle by regulating the extracellular matrix. Activated satellite cells repressed fibroblast collagen production via extracellular vesicle (EV) delivery of miR-206, revealing a previously unrecognized function of satellite cells, in addition to providing a mechanism through which satellite cells communicate with other cells within muscle. We now have in vivo single-cell (sc)RNA-seq evidence from Pax7-tdT reporter mice of a satellite cell intercellular communication network in which satellite cells communicate with FAPs/fibrogenic cells and endothelial cells during hypertrophic growth. Aim 1 will test the hypothesis that aging negatively impacts this communication network inhibiting proper remodeling of the extracellular matrix thereby inhibiting hypertrophy. We have also developed a novel mouse model that allows us to simultaneously deplete satellite cells and label resident myonuclei, Aim 2 will use this model and single-myonuclear (smn)RNA-seq to characterize age-dependent changes in the resident myonuclear transcriptome and identify mechanisms that enable short term hypertrophy in the absence of satellite cells in adult mice, which is lost in old age. Aim 3 will use an additional newly developed reporter mouse that enables specific and stable labeling of satellite cell nuclei to determine how aging alters the satellite cell-derived myonuclear transcriptome in response to a hypertrophic stimulus. We hypothesize that in aged muscle, satellite cell-derived myonuclei have altered transcriptional output that does not promote a hypertrophic response, and that impaired fusion of satellite cells or defective satellite cells may negatively impact resident myonuclear transcriptional activity contributing to impaired growth. Defining fusion-dependent and -independent roles of satellite cells and age-associated changes that negatively impact muscle adaptability will identify potential new targets to promote skeletal muscle growth in the face of inactivity, during aging and in the face of muscle wasting diseases.

Summary A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and function. Recent estimates indicate that up to one-third of the elderly population may suffer from frailty. Frailty often leads to loss of functional independence and is among the leading risk factors for falls in the elderly. In fact, as life expectancy continues to increase, the maintenance of muscle strength is an important factor that contributes to a higher quality of life. Although the etiology of sarcopenia remains to be determined, proposed contributing factors include the loss of innervation and motor unit decline, oxidative stress, aberrant autophagy and inflammation. There is emerging evidence for the existence of a gut-skeletal muscle axis. On the basis of this connection, there has been much speculation that dysbiosis of the gut microbiota might have a role in the development of sarcopenia. While plausible mechanisms have been proposed through which dysbiosis might contribute to the development of sarcopenia, there remains a paucity of direct evidence to support such mechanisms. The purpose of this exploratory grant is to test the hypothesis that dysbiosis of the gut microbiota promotes sarcopenia by inducing a state of anabolic resistance in skeletal muscle through TLR4 hyperactivation of mTORC1 signaling. To test this hypothesis, the following aims will be pursued: Aim 1: Determine if the gut microbiota from aged mice can induce sarcopenia in adult mice; Aim 2: Determine if the gut microbiota from adult mice can rescue sarcopenia in old mice. If the results of the proposed experiments provide evidence to support the hypothesis, a future R01 application will seek to rigorously test the proposed mechanism by determining if a skeletal muscle-specific TLR4 knockout prevents sarcopenia despite low-grade systemic inflammation induced by gut microbiota dysbiosis.