Karyn Esser - US grants

DESCRIPTION (Adapted from the applicant's abstract):Both neural and weight-bearing activities have been shown to be important physiological factors regulating slow skeletal muscle phenotype in the adult animal. While these phenotypic changes have been well characterized, there is not much known about the molecular mechanisms through which neuromuscular activity regulates the slow contractile protein isoform genes. The long-term objectives of this project are to understand mechanisms through which contractile protein gene families are regulated in the acquisition and maintenance of diverse skeletal muscle fiber types. Previous work from this lab has shown that - 270 bp of the myosin light chain 2 slow (MLC2slow) promoter is sufficient to direct both slow nerve and mechanical load dependent expression. Further analyses have identified that the CACC and MEF2 sites within this 270 bp region cooperate in regulating transcription in response to slow innervation. The specific aims of this proposal are: 1) to identify the proteins that contribute to nerve and mechanical load dependent transcription factor complex formation at the CACC and MEF2 sites of the MLC2slow promoter; 2) to determine mechanisms by which neural and mechanical activity regulate the transcriptional activity of the factors identified in Aim 1; and 3) To determine the mechanisms by which altered cellular calcium modulates transcription factor functions in vitro and in vivo. This proposal combines both in vitro and in vivo approaches to identify the critical factors and pathways responsible for physiological regulation of the MLC2slow promoter. This work has broad application to basic and applied areas of biomedical and health science fields. At the basic science level, new insight will be gained into mechanisms by which DNA elements and factors regulate a specific contractile protein isoform gene. Contractile proteins are a major topic of investigation not only for muscle biology, but their function and regulation are also implicated in maintenance of cell architecture and morphology, cell cycle events, and cell transformation/cancer. At the more applied level, because of the common occurrence of muscle regeneration in humans, this study will have wide clinical application. Muscle regeneration does occur following muscle transplant surgery for correction of facial paralysis; following muscle damage due to mechanical, thermal, or metabolic stress; as well as its association with dystrophic muscle pathologies.

DESCRIPTION (Applicant's abstract): It has been known for many years that high resistance exercise results in the hypertrophy of skeletal muscle, however, to date very little is known about the signaling mechanisms regulating this growth response. Studies of exercise-induced hypertrophy in mammals have shown that an early response to one bout of exercise is a transient increase in protein synthesis followed by subsequent increases in muscle specific mRNAs. This pattern of change suggests a model in which the transient increase in protein synthesis produces specific factors important for muscle growth. The overall aims of this project are to elucidate the upstream signaling mechanism(s) necessary for protein synthesis changes following resistance exercise and to determine the role of translational activation in the development of skeletal muscle hypertrophy. We have modified an in vivo model to study the regulation of exercise-stimulated protein synthesis. With this model, the phosphorylation of p70s6k was identified a an acute marker of the hypertrophic growth response. This provides a critical starting point from which experiments have been designed to delineate the growth- related signaling pathways stimulated by exercise. We also have found that p70s6k is activated following stretch-induced growth of myotubes in vitro. The experiments proposed rely on the use of in vitro stretch system to screen for the critical factors and pathways activated. These factors and pathways will subsequently be tested for their physiological significance in vivo. The findings from this project are important for the basic understanding of how striated muscle cells regulate their size in response to increased loading. In addition, the results from these studies have tremendous application for the fields of aging and rehabilitation. Age-associated muscle atrophy is a debilitating condition that can decrease the quality of life as well as limit independent living for the elderly. Understanding the factors and pathways that regulate the growth response of skeletal muscle will be important for designing therapeutic treatments (pharmacological or exercise based) to attenuate or reverse losses in muscle mass.

DESCRIPTION (provided by applicant): Circadian rhythms have long been known to influence behavioral and biological processes such as physical activity and feeding behavior. The fundamental importance of this system, which works to link physiology with the day/night cycle, is underscored by its presence in every known species. Recent studies have identified that most cell types, including skeletal muscle, display circadian oscillations in gene expression and function. While the central clock, the superchiasmatic nucleus (SCN), is considered the master regulator, growing evidence has defined a level of autonomy for the peripheral clocks. In the case of skeletal muscle, there is very little known about circadian rhythms with only a few studies in humans that have demonstrated maximum force generation varies with time of day. There are, however, several lines of evidence that link regulation (entrainment) of core circadian oscillators to the energy and force production driven by locomotor activity. These observations clearly suggest a potentially significant role for the limb musculature to circadian rhythm biology. One of the fundamental core circadian rhythm genes, Bmall (Brain muscle arnt like 1/MOP3), is characterized by its abundant expression in skeletal muscle. Interestingly, mice in which Bmall has been ablated exhibit a significant reduction in voluntary wheel running activity by over 60% as well as the expected loss of circadian behavior (7). Recent results from microarray experiments have identified that Bmall mRNA is significantly increased in skeletal muscle of both humans and rats at 6 hours following an acute bout of high resistance exercise (8) (Zambon, UCSF, personal communication). The studies outlined in this R21 proposal, are designed to test the following hypotheses: 1) skeletal muscle, independent of innervation, displays circadian periodicity in function and 2) appropriate expression of the circadian rhythm genes, in particular Bmall, is necessary for normal skeletal muscle function and phenotype. Results from these studies will be critical in providing the foundation for future studies on the complex interaction between skeletal muscle circadian gene expression, muscle function and physical activity behaviors.

DESCRIPTION (provided by applicant): Growth and maintenance of skeletal muscle mass is critical for long-term health and quality of life. Muscle is important for mobility but it is also crucial for metabolic health because of its contribution to glucose uptake and fat metabolism. Our lab and others have demonstrated that signaling through the mammalian target of rapamycin (mTOR) is necessary for skeletal muscle growth but the molecular links between signal input and downstream function are far from understood. In our previous studies we showed that a) prolonged activation of mTOR signaling is specific to growth inducing contractions, b) mechanical strain activates mTOR signaling in skeletal muscle but not in non-muscle cells, c) strain-induced mTOR signaling is independent of the IGFl/insulin/PI3 kinase signaling pathways, and d) cell cycle genes, such as cyclin D1 and c-myc, are induced during mTOR dependent growth and they likely function, in the differentiated muscle cell, to induce ribosomal biogenesis/protein synthesis. The overall goal of this research is to use in vitro and in vivo models of skeletal muscle growth with molecular tools to identify the necessary upstream regulators and downstream effectors of mTOR. I) mechanical strain acts synergistically with IGF1 to prolong activation of mTOR signaling and II) mTOR kinase activity is necessary for translational-regulation of critical growth mRNAs including cyclin D1 and c- myc. These hypotheses will be tested by the following specific aims: Specific Aim 1. To determine the molecular signals by which mechanical strain regulates mTOR signaling. Specific Aim 2. To demonstrate whether mechanical signaling acts synergistically with IGF1 to regulate growth-related mTOR function. Specific Aim 3. Specific Aim 3. To determine whether the kinase activity of mTOR is necessary for growth, protein synthesis and increased c-myc and cyclin D1 protein levels in response to mechanical overload in vivo. The results of these studies will provide novel insight for the field of mTOR regulation and the role of mechanical strain in the growth process of skeletal muscle. The clinical implications of this work are also significant and will contribute to development of strategies to attenuate or ameliorate muscle atrophy associated with disuse, aging, bed rest and cachexia.

Project Summary: Circadian rhythms have long been known to influence behavioral and biological processes such as physical activity and feeding behavior. The fundamental importance of this system, which works to link physiology with the day/night cycle, is underscored by its presence in every known species and the growing evidence linking alterations in core clock function and diseases such as cancer, diabetes and mental health. Recent studies from our laboratory have shown that mutations of the canonical circadian genes, Clock and Bmal1, dramatically disrupt muscle function. This is the first genetic evidence linking skeletal muscle function to circadian rhythms. Expression profiling studies of skeletal muscle over 48 hours identified that MyoD is a circadian gene. Molecular experiments went on to demonstrate that the MyoD promoter is directly bound and regulated by the core clock factors, CLOCK and BMAL1. The studies outlined in this proposal combine molecular, cell biological and biophysical approaches to understand the mechanisms by which the core clock mechanism is regulated in skeletal muscle and how loss of this rhythm leads to altered MyoD expression and loss of muscle function. The hypotheses for this proposal are 1) Synchronization of core clock gene expression in skeletal muscle requires intact innervation, 2) Muscle specific mutations of Bmal1 or Clock will be sufficient to cause arrhythmic MyoD expression and muscle dysfunction 3) Loss of maximal force capacity/specific tension in muscle of circadian clock- compromised mice is due to decreased actin:myosin interaction resulting from disruption of sarcomeric structure and/or altered stoichiometry of myofilament proteins. The results of the experiments outlined in this proposal have significant implications for maintenance of muscle with an impact on our understanding and treatment of sarcopenia, muscle wasting/cachexia and problems associated with metabolic diseases such as insulin resistance. In addition, our understanding of circadian regulation of skeletal muscle will have likely applications for rehabilitation therapies for spinal cord injury patients and people that are exposed to prolonged periods of bedrest. Project Narrative The results of the proposed work will determine the role of the biological clock in maintaining normal skeletal muscle structure and function. The findings have significant implications for maintenance of muscle with an impact on our understanding and treatment of sarcopenia, muscle wasting/cachexia and problems associated with metabolic diseases such as insulin resistance. In addition, our understanding of circadian regulation of skeletal muscle will have likely applications for rehabilitation therapies for spinal cord injury patients and people that are exposed to prolonged periods of bedrest.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area 15: Translational Science and specific Challenge Topic 15-ES-101: Effects of environmental exposures on phenotypic outcomes using non-human models. Approximately 8.6 million Americans perform shift work, which is associated with increased risk of cardiovascular and cardiopulmonary diseases. Light pollution is one of the environmental conditions that is suggested to be a contributor to the increased pathologies in shift workers. In 2007 the NIEHS released a report on light pollution noting;"that the dramatic increases in chronic diseases in modern society maybe associated with the altered patterns of light and dark". One of the key physiological targets of altered patterns of environmental lighting is the circadian timing system. There is growing recognition that the increased pathologies seen in shift workers could arise from misalignment between the molecular circadian timing system within tissues/organs and the altered environmental time cue due to disrupted light exposure. The goal of the projects described in this Challenge Topic application will use targeted tissue specific disruption of a core circadian gene, Bmal1, with controlled manipulation of environmental light cues to determine the interaction between genetic and environmental factors in the progression of cardiopulmonary disease. Analyses will include use of in vivo telemetry and echocardiography to provide longitudinal data on systemic disease progression. In addition, experiments will be performed that will provide mechanistic insight using molecular, cellular and biochemical approaches. The overall hypothesis for this project is that targeted deletion of Bmal1 in muscle tissues (heart or smooth or skeletal) will weaken the animal's ability to handle light pollution and will be associated with a more rapid and profound progression to cardiopulmonary diseases. This is a novel area of research for this team of established investigators in muscle biology and cardiopulmonary disease. Their combined expertise and prior history of successful collaboration in the areas of circadian rhythms, cardiac, smooth and skeletal muscle biology will allow for rapid progression on this high priority research area. At the end of this two-year project we are confident that will have obtained significant new data regarding the interaction between the molecular clock function in cardiopulmonary tissues and environmental light challenges and their contribution to disease progression. Approximately 8.6 million Americans perform shift work, which is associated with increased risk of cardiovascular and cardiopulmonary diseases. As reported in 2007 by NIEHS, light pollution is one of the environmental conditions that is suggested to be a contributor to the increased pathologies in shift workers. The goal of the projects in this application will determine the interaction between altered circadian genes with environmental factors in the progression of cardiopulmonary disease.

DESCRIPTION (provided by applicant): As the house-building macromolecule of the cell, ribosome biogenesis is essential for cell growth. Despite this central role in cell growth, there remains a fundamental gap in our understanding of the role of ribosome biogenesis in adult skeletal muscle hypertrophy. Studies from our laboratory have provided evidence which supports a role for increased ribosome biogenesis in skeletal muscle hypertrophy. The current proposal will begin to directly examine the importance of ribosome biogenesis to muscle hypertrophy by testing the hypothesis that -catenin is necessary for muscle hypertrophy by increasing protein synthesis through c-myc activation of ribosome biogenesis. To conditionally, manipulate -catenin or c-myc gene expression in adult skeletal muscle we generated the HSA-MerCreMer mouse. Aim 1 will determine if ?-catenin expression is necessary for skeletal muscle hypertrophy using a mechanical overload model of the plantaris muscle following catenin gene inactivation. Aim 2 will determine if increased expression of ?-catenin is sufficient to stimulate skeletal muscle hypertrophy. ?-catenin will be over-expressed in adult skeletal muscle by using the HSA- MerCreMer strain to generate a stabilized form of ?-catenin. Aim 3 will determine if c-myc expression is necessary for skeletal muscle hypertrophy following the conditional inactivation of c-myc in adult skeletal muscle using the HSA-MerCreMer strain. The effect of gene inactivation on the hypertrophic response will be assessed by measuring morphometric (muscle weight, fiber CSA), biochemical (total protein, RNA and DNA), molecular (Western blot, RT-PCR, promoter analysis, chromatin immunoprecipitation (ChIP) and electrophorectic mobility shift assay (EMSA)) and metabolic (rates of protein synthesis and degradation) variables. The results of the proposed studies are expected to have important clinical implications by identifying new molecular targets for promoting skeletal muscle protein synthesis and hypertrophy. In the long- term, the ability to manipulate ribosome biogenesis represents a promising novel strategy to attenuate or ameliorate muscle atrophy associated with aging, bed rest and cachexia.

DESCRIPTION (provided by applicant): This is a revised application to our parent grant Beta-catenin regulation of skeletal muscle hypertrophy being submitted in response to RFA-AR-13-003, NIAMS Building Interdisciplinary Research Team (BIRT) Revision Awards. We have established an interdisciplinary research team with Dr. Dasarathy, a transplant hepatologist with a joint appointment in the Department of Pathobiology at the Lerner Research Institute in the Cleveland Clinic. In addition to his clinical duties, Dr. Dasarathy heads an active research program investigating the molecular and metabolic mechanisms underlying skeletal muscle sarcopenia induced by cirrhosis. This work has found that hyperammonemia caused by cirrhosis leads to sarcopenia as the result of increased myostatin expression and decreased protein synthesis. Preliminary data in C2C12 myotubes showed that over- expression of ¿-catenin was able to block the increase in myostatin expression in response to ammonium. Based on this preliminary data, we propose in this revision to test the hypothesis that over-expression of ¿-catenin will prevent the deleterious effects (decreased protein synthesis, loss of muscle mass) caused by hyperammonemia-induced myostatin expression. To test this hypothesis, the following aim will be pursued: Determine if over-expression of ¿-catenin is able to protect skeletal muscle against hyperammonemia. Both in vitro and in vivo experiments will be performed to determine if over-expression of ¿-catenin is capable of mitigating the deleterious effects of myostatin expression induced by ammonium. The proposed in vitro and in vivo studies are expected to demonstrate that over-expression of ¿-catenin is capable of rescuing the sarcopenic phenotype by repressing myostatin expression. These proposed experiments extend the scope of the original proposal beyond the regulation of ribosome biogenesis by ¿-catenin to examine the ability of ¿-catenin to prevent cirrhosis-induced sarcopenia. The notion that ¿-catenin may provide a mechanism for rescuing the loss of muscle mass associated with cirrhosis has significant clinical implication given the recent identification of small molecules capable of activating ¿-catenin signaling. In addition, the data generated under this award are expected to serve as the basis for an R01 grant application in response to PA-12-208 Functions of Skeletal Muscle beyond Contractions which seeks to promote research that leads to novel strategies to protect or treat common diseases and conditions.

DESCRIPTION (provided by applicant): Skeletal muscle weakness has long been known to contribute to morbidity and mortality in aging and other diseases. Single fiber studies from different models of weakness in humans and rodents have demonstrated reduced force implicating alterations in myofilament protein expression, post-translational modifications and sarcomere organization. Our recent work in the emerging area of circadian rhythms and the molecular clock in skeletal muscle holds potential to provide insight into mechanisms of weakness. Most recently we generated an inducible line of mice in which Bmal1 is deleted only in adult skeletal muscle following tamoxifen treatment (iMSBmal1-/-). We observed progressive declines in cage activity and voluntary wheel activity and we determined that the muscles were weak with significant declines in maximum force and passive tension. To begin to identify the molecular mechanism(s) underlying weakness in this model, we have identified circadian expression of transcription factors important for muscle (Srf, Sox6 and Tead1) and we found that expression of these genes was disrupted in the muscle of iMSBmal1-/- mice. These observations support our hypothesis that the molecular clock in skeletal muscle directly regulates expression of a network of important transcription factors (muscle clock controlled genes) and when the molecular clock is disrupted, this leads to downstream effects on sarcomere gene expression, sarcomere structure and muscle mechanical function. In addition to work on the molecular clock, my lab and others have demonstrated that scheduled physical activity can function as an environmental non-photic time cue for the skeletal muscle molecular clock. This discovery highlights a new mechanism for physical activity and provides the basis for our second hypothesis: Time of exercise will act as a therapeutic intervention to slow the progression of muscle weakness in aging. These two hypotheses will be tested in the following three specific aims. Specific Aim 1: To determine whether the myogenic transcription factors, Srf, Sox6, Tead1 are direct molecular clock controlled genes in skeletal muscle. Specific Aim 2: To determine the sarcomeric changes through which active and passive tension is reduced in skeletal muscle fibers of iSMBmal1-/- mice. Specific Aim 3: To determine whether time of exercise modifies the rate of progression of muscle weakness with aging. The results of these studies will provide new insight into mechanisms that link disrupted circadian rhythms and muscle weakness. Since aging and many chronic diseases are known to disrupt molecular clock function these findings may also provide novel new targets for therapeutic strategies.

Abstract Exercise is a powerful and pleiotropic physiological stimulus that helps prevent many chronic diseases and is used as a therapeutic for disease. While the beneficial effects of exercise are extensively acknowledged there is still very little understood about the molecular transducers of the systems-wide effects. The goal of this University of Florida Molecular Transducers of Physical Activity Preclinical Animal Study Sites application (UF PASS) is to conduct experiments in animals that will provide tissues/blood (i.e. biospecimens) to the Chemical Analysis Sites for identification of molecular transducers induced by defined models of physical activity from tissues that cannot be obtained from humans as well as to conduct mechanistic studies that can support screening of novel transducers to quickly move the field forward. In Phase 1, UF PASS proposes to collect biospecimens for the Chemical Analysis sites following endurance (run-training) or resistance exercise protocols on male and female Fischer 344xBrown Norway rats (F344-BN) at three different ages. To better capture the dynamics of the exercise/adaptation responses we propose to: 1) Collect biospecimens at 5 selected timepoints following an acute bout of exercise on naïve and trained rats; 2) Collect biospecimens following short duration training (after 5 bouts) and 3) Collect biospecimens following long-term (8 weeks) training. For Phase 2, our hypothesis is that factors released from muscle (i.e. myokines) are the molecular transducers that function throughout the system to improve the well-established stress tolerance. The goal of these studies will be to employ high throughput screening technologies to test up to 1500 myokines. We will then use secondary screening techniques to test 100 candidates from which we will select up to 3 candidates for in vivo testing. The results of the experiments in Aim 3 will provide molecular evidence identifying a set of transducers, released from muscle, that are necessary for exercise induced systemic health. The goals of the UF PASS will be pursued by the following Specific Aims: Specific Aim 1: Center Coordination Phase. Specific Aim 2: Phase 1 Studies. To perform endurance and resistance exercise using male and female F344BN rats at 3-4, 16-18, and 27-29 mo. Specific Aim 3: Phase 2 Studies. The goal in Aim 3 is to test myokines as the exercise transducers for improved stress tolerance.

Abstract: This administrative supplement is submitted in response to Notice Number: NOT-AG-18-008: Alzheimer's Disease and its related Dementias (AD/ADRD)-focused Administrative supplements for NIH grants that are not focused on Alzheimer's disease. This supplement is outlines experiments to extend the parent R01, Molecular clock and skeletal muscle weakness (R01 AR066082) to determine the impact of disruption of the skeletal muscle clock on Alzheimer?s disease onset and progression. Studies of skeletal muscle have not been the primary focus of Alzheimer?s Disease research, however, there are clinical studies in the last 10 years that have observed loss of skeletal muscle mass/sarcopenia occurs at a faster rate in Alzheimer?s disease patients and this is associated with brain atrophy and diminished cognitive performance. There are also several studies with animal models of AD that demonstrate changes in skeletal muscle strength and metabolism at stages prior to the appearance of amyloid plaques in the brain. My lab has generated preliminary data that shows that skeletal muscles of our mice, mBmal1 cKO, exhibit accelerated aging phenotype with changes in expression of the secreted glycoproteins, which are known to inhibit protein aggregation of misfolded pathological proteins that are associated with AD. We propose to test the hypothesis that muscle specific loss of Bmal1, will lead to accelerated onset and progression of neurodegeneration in a mouse model of Alzheimer?s disease. In addition, we will also test the provocative concept that loss of Bmal1 in skeletal muscle is sufficient to induce AD-related changes in the brain. The Specific Aim to be tested is: Aim 1) To determine whether loss of Bmal1 in adult skeletal muscle will alter proteostatic networks in the hippocampus and cortex and modulate the seeding and evolution of AD pathologies. Collectively, these studies will provide new insight into the role of skeletal muscle and skeletal muscle homeostasis on the evolution of Alzheimer?s Disease pathologies.

Summary: The overall objectives of this proposal are to 1) define the genomic and transcriptomic mechanisms by which the cardiomyocyte clock regulates ion channels that contribute to cardiac excitability; and 2) disrupt the cardiomyocyte clock to link changes in circadian-ordered gene expression with electrophysiological properties of atrial and ventricular cardiomyocytes. The outcomes will address significant gaps in our understanding for how the myocardial circadian clock regulates the expression of key cardiac ion channels and how abnormal cardiac clock function contributes to arrhythmia vulnerability. The mechanism regulating circadian timing, the molecular clock, exists in virtually all cell types in the body. A critical function of the molecular clock is to link time of day with a large-scale transcriptional program to support cellular homeostasis To date, our labs have used an inducible cardiomyocyte specific mouse model to knock out the core clock gene, Bmal1 (iCS?Bmal1). These studies showed that disruption of the myocardial clock is sufficient to decrease ventricular K+ and Na+ channel gene expression, disrupt current levels, disrupt cardiac excitability, and increase arrhythmia susceptibility. These studies establish a critical role for the cardiomyocyte clock, independent of the central clock, in regulating the expression of different families of ion channel genes that impact the ionic balance needed for normal excitability. One goal of this project is to utilize large scale genomic and transcriptomic approaches with our mouse model system to define the circadian clock dependent control of temporal gene expression in both atrial and ventricular tissues. To address abnormal circadian clock function, our lab has used different models of circadian disruption, such as chronic phase advance or time restricted feeding to test links between circadian disruption and arrhythmia vulnerability in mouse models. We have found that disrupting either light or feeding time cues is sufficient to induce pathological changes in cardiac rhythms in normal mice and to accelerate sudden cardiac death in a genetic mouse model of arrhythmia susceptibility. These studies support our premise that disruption of day- night rhythms through environmental factors leads to altered myocardial clock function with outcomes that include modified ion channel expression, cardiac excitability and arrhythmia vulnerability. The aims of this proposal are designed to test the following hypotheses: 1) The molecular clocks in both atrial and ventricular cardiomyocytes are necessary to direct daily chromatin accessibility and transcriptional output including expression of key ion channel and ion channel regulatory genes. 2) Chronic disruption of the cardiomyocyte clock using altered time of feeding is sufficient to cause dysregulation of the cardiac clock resulting in an imbalance in cardiac ion channel expression and currents leading to altered excitability and increased arrhythmia vulnerability.

Abstract: The purpose of this project is to provide research and career development training for Silvana Sidhom, a current PhD student at the University of Florida. This administrative supplement in response to PA- 21-071: ?Research Supplements to Promote Diversity in Health-Related Research (Admin Supp)? will facilitate career development for the candidate and as well as contribute to the mission of the parent project ? to define molecular targets downstream of the circadian clock that modulate muscle structure with implications for strength. The primary objectives of Ms. Sidhom?s proposed research will be to work on experiments related to Aim 2 of the parent grant. First, she will learn to use confocal imaging approaches with sarcomeric antibodies to define changes in the M-line and Z-line structural elements of the sarcomere from the control and muscle specific Bmal1 KO mice. Second, she will use AAV approaches to restore expression of selected sarcomeric genes, such as Tcap, to test if that is sufficient to restore structure and muscle force. Ms. Sidhom will work actively with Collin Douglas, another PhD student working on the parent grant as well as Dr. Christopher Wolff, a postdoctoral associate on this project. Dr. Esser, the PI of the R01, will be the primary mentor for Ms. Sidhom within the College of Medicine at the University of Florida. We have established her committee and this includes: Drs. Russ Hepple, Christiaan Leeuwenburgh and Richard Dunn. They are enthusiastic about Ms. Sidhom as a student and provide appropriate expertise in skeletal muscle and imaging approaches. Silvana has completed her first year of required courses so the committee will work with Ms. Sidhom to outlined a series of didactic courses, including the skeletal muscle course taught each fall. The committee will also help guide Silvana with experiential training activities that will equip her with the necessary skills to progress as an independent scientist and to fulfill the NIH mission to advance the careers of a diverse population of scientists. The project is directly related to Aim 2 of the parent grant: Specific Aim 2: To test the clock controlled genes, Rbm20 and/or Tcap, for their roles in sarcomere structure and muscle function. This aim will use AAV delivery of Tcap and/or Rbm20 in Bmal1 deficient muscle to test their contribution to sarcomere structure, myofibrillar protein composition and muscle function. Sarcomere structural elements will be obtained by super-resolution microscopy with deconvolution techniques with antibodies to define 2D and 3D properties including sarcomere length, thick filament centrality, M-band and Z line integrity. Deep RNA-sequencing will be performed on the Rbm20 muscles to define splicing changes. Functional outcomes will include ex vivo measures to test the impact of structural changes on both passive/stiffness and active/max force and rate of contraction properties.

Abstract: This administrative supplement is submitted in response to Notice Number: NOT-AG-20-034: Alzheimer's Disease Administrative supplements for NIH grants. This supplement outlines experiments to extend the parent U01 (UF PASS: Regulation of exercise transducers) to pursue the interaction between exercise, skeletal muscle health and progression of pathology in a mouse model of Alzheimer?s disease (AD). The parent award is part of the MoTrPAC consortium that combines well defined exercise interventions with multi-omics analysis to develop a map of molecular transducers that link exercise to systemic health. For this administrative supplement we will use a defined mouse model of AD with a treadmill training protocol based on the protocol used for MoTrPAC. We will also be in a position to analyze our outcomes data in the context of the larger multiomics data available from MoTrPAC to expand our understanding of potential cross tissue interpretations. A major challenge in the field of Alzheimer?s disease (AD) is the poor understanding of how tau pathology promotes disease. Consequently, therapeutic interventions are ineffective and clinical trials for AD have failed. The working model for this supplement is that exercise mitigates the onset and progression of AD tau pathology through impacting skeletal muscle health as well as direct effects on the brain. The rationale for this model comes from two complementary but not fully explored sub-fields: ?AD and muscle/sarcopenia? and ?AD and exercise?. Several clinical studies have reported that muscle weakness and loss of muscle mass occurs at a faster rate in AD patients. These muscle changes in the patients are also associated with brain atrophy and diminished cognitive performance. In addition, several studies using animal models of AD have demonstrated changes in skeletal muscle metabolism at times prior to the appearance of AD pathology in the brain. These observations are consistent with our preliminary data that found significant muscle weakness in a tauopathy mouse model of AD prior to overt signs of neurodegeneration. There are also a large number of clinical and preclinical studies showing that exercise training, primarily endurance exercise, has a positive impact on the onset and progression of AD. Thus, we propose to integrate the concepts of exercise, muscle health and brain health to define molecules and pathways that attenuate the onset and progression of AD tau pathology in the brain. We propose the following Specific Aims. Aim 1) To determine the molecular, histological and phenotypic changes in skeletal muscle and brain in an acquired tauopathy model of AD in mice. Aim 2) To use a running exercise intervention with the AD-tauopathy mouse model to identify molecular sites through which tau pathology is attenuated.

We have shown that disruption of the muscle circadian clock mechanism through loss of the core clock gene, Bmal1, is sufficient to induce significant muscle weakness and surprisingly, increased mortality. Based on these findings, the overall objective of this grant is to pursue the fundamental understanding of the role of the muscle circadian clock in regulating a daily program of gene expression and how clock disruption leads to significant muscle weakness and diminished systemic health. We found that MyoD1 can modulate expression of the core clock gene, Bmal1 making it a bona fide tissue- specific circadian clock modifier1. We have also determined that MyoD1 and CLOCK:BMAL1 share peak binding at over 3000 sites across the muscle genome. These new findings provide support for our studies to define the mechanism(s) through which MyoD1 modulates the network properties of the clock mechanism as well as understanding the role of MyoD1 as a clock co-factor in the daily genomic and transcriptomic landscape in adult muscle. Downstream from MyoD1 and the clock factors, my lab has identified two muscle specific genes, Rbm20 and Tcap, that we propose link clock disruption with muscle weakness. Loss of muscle Bmal1, results in significant decreases in Rbm20 and Tcap expression and we find changes in sarcomere structure including variability of sarcomere length, distortions in M and Z lines and altered myofilament orientation. Lastly, the global Bmal1 knock out mouse, Bmal1KO, has been used as a model of advanced aging as it exhibits significant aging-like pathologies and has a median lifespan of 37wks. In preliminary experiments using this global Bmal1 KO mouse we rescued Bmal1 in skeletal muscles using an AAV vector with a muscle specific promoter. We found that this was sufficient to significantly improve muscle strength but also significantly extended lifespan. These are complementary to our findings of increased mortality with loss of muscle Bmal1 and demonstrate that rescuing Bmal1 only in skeletal muscle improves systemic health. In addition, with aging and many chronic diseases exhibiting muscle clock disruption, these results suggest that targeting the muscle clock mechanism holds potential as a translational strategy. We propose to test the following three specific aims: Specific Aim 1: To define the roles of MyoD1 within the core clock mechanism and as a co-factor for the daily transcriptomic landscape in skeletal muscle. Specific Aim 2: To test the clock controlled genes, Rbm20 and/or Tcap, for their roles in sarcomere structure and muscle function. Specific Aim 3: To determine the skeletal muscle specific changes required for improved lifespan in the Bmal1 KO mouse.

Abstract: The purpose of this Research Supplement is to Promote Diversity in Health-Related Research through support of the career development training for Bryan Alava, a current PhD student at the University of Florida. This administrative supplement will facilitate career development for the candidate and as well as contribute to the mission of the parent project - to discover novel exercise induced health related outcomes. The parent award is part of the MoTrPAC consortium that combines well defined exercise interventions with multi-omics analysis to develop a map of molecular transducers that link exercise to systemic health. To date, the consortium is analyzing the multi-omics data across 17 different tissues collected following a defined treadmill exercise paradigm as part of the preclinical studies. The primary objectives of Mr. Alava?s proposed research are two-fold: First to use a defined Alzheimer?s Disease (AD) mouse model to characterize changes in skeletal muscle function and blood markers of inflammation and metabolism. Second, once characterized we will use this model to test a treadmill training protocol, based on the protocol used for MoTrPAC, for efficacy in delaying the progression of AD. We will also be in a position to access the larger multiomics data available from MoTrPAC to expand potential molecular targets of exercise and AD pathology. Dr. Esser, the PI of the U01, will be one of two primary mentors for Mr. Alava. Dr. Jose Abisambra is the other co-mentor as his lab provides the AD mouse model and expertise in the analysis of brain pathologies. Drs. Esser and Abisambra are already official co-mentors for Bryan Alava within the Ph.D. program in the Department of Physiology and Functional Genomics within the College of Medicine at the University of Florida. We have outlined a series of didactic and experiential training activities that will equip Mr. Alava with the necessary skills to progress as an independent scientist and to fulfill the NIH mission to advance the careers of a diverse population of scientists.