PROJECT SUMMARY/ABSTRACT Skeletal muscle mass declines with age and simultaneously loses the ability to adapt to stressors. Reduced muscle mass and function associated with aging ultimately leads to a loss of independence and mortality, costing billions of dollars to the US economy every year. Understanding what contributes to muscle mass loss with age and what mediates the ability to enhance muscle mass with exercise or alternative therapeutics is of critical importance for developing effective countermeasures. The study of epigenetics, and specifically DNA methylation status has become increasingly popular in recent years since this level of regulation heavily dictates what genes are expressed and for how long. Skeletal muscle is multi-nucleated and ~50% of nuclei in the muscle compartment are myonuclei. Little can be inferred about DNA methylation in muscle fibers without the ability to specifically isolate myonuclei. We recently developed a genetically modified mouse model for labeling skeletal muscle myonuclei and a workflow for purifying these labeled myonuclei for downstream epigenetic applications. We also developed a translatable and reversible murine model of progressive weighted wheel running (PoWeR) that elicits hypertrophy in muscles of different fiber type composition and function. Using these novel tools, the purpose of this proposal is to provide the first muscle type-specific insight into the epigenetic regulation of skeletal muscle mass with aging, and determine whether early life stimuli affect muscle adaptability later in life. I will profile myonuclear DNA using reduced representation bisulfite sequencing in order to explore global methylation status, and focus in on ribosomeal DNA (rDNA) methylation since ribosome biogenesis is strongly implicated in skeletal muscle mass regulation. In the mentored phase of the grant, I will learn the requisite epigenetic techniques for studying muscle mass regulation by evaluating myonuclear epigenetics during hypertrophy (PoWeR) and atrophy (hindlimb suspension). With these newly acquired skills, I will profile myonuclear DNA from oxidative and glycolytic muscles during the independent phase to provide a comprehensive epigenetic map of muscle aging. I will also PoWeR train mice early in life to determine whether previous hypertrophy affects adaptability to that same stimulus later in life with the aim of identifying epigenetic cues for improving muscle adaptability in old age. My global hypotheses are that hypertrophy will be associated with muscle type-specific global and rDNA hypomethylation, while unloading and aging will be characterized by muscle type-specific hypermethylation. I also hypothesize that early life exercise will be associated with rDNA hypomethylation later in life, which will associate with restored hypertrophic adaptability in old age. The experiments in this proposal will provide a foundation for the development of epigenetic-based therapeutics aimed at skeletal muscle rejuvenation with aging. Furthermore, the training opportunity afforded by this project will facilitate my success as an independent researcher focused on the epigenetics of muscle mass regulation during aging in skeletal muscle.

PROJECT NARRATIVE Skeletal muscle mass declines with age and simultaneously loses the ability to adapt to exercise, which collaboratively leads to a loss of independence and mortality and costs billions of dollars to the US economy every year. A major determinant of phenotype and plasticity is gene transcription that is in large part controlled by DNA methylation, but essentially nothing is known about the epigenetic control of skeletal muscle mass regulation with aging since isolating skeletal muscle nuclei has presented a significant challenge. The techniques developed for this project overcome this roadblock, and mapping the myonuclear methylome in muscles of different type and function in the context of exercise and aging will provide foundational insight for combatting age-mediated skeletal muscle mass loss and dysfunction.