Myonuclear Epigenetics of Skeletal Muscle Mass Regulation with Age

项目概要/摘要 骨骼肌质量随着年龄的增长而下降，同时失去了适应压力的能力。减少 与衰老相关的肌肉质量和功能最终导致独立性的丧失和死亡， 每年给美国经济造成数十亿美元的损失。了解什么有助于肌肉质量损失 随着年龄的增长，什么介导的能力，以提高肌肉质量与运动或替代疗法是 制定有效的对策至关重要。表观遗传学研究，特别是DNA 近年来，甲基化状态变得越来越普遍，因为这种水平的调节严重影响了细胞的生长。 决定了哪些基因被表达以及表达的时间。骨骼肌是多核的， 肌间室是肌核。在没有DNA甲基化的情况下， 特异性分离肌细胞核的能力。我们最近开发了一种转基因小鼠模型， 标记骨骼肌肌细胞核和纯化这些标记的肌细胞核用于下游的工作流程 表观遗传学的应用我们还开发了一种可翻译的和可逆的小鼠模型， 负重轮跑（PoWeR），其在不同纤维类型组成的肌肉中引起肥大， 功能使用这些新的工具，这项建议的目的是提供第一个肌肉类型的具体见解 研究骨骼肌质量随年龄增长的表观遗传调节，并确定早期生活刺激是否影响 肌肉的适应能力。我会用还原亚硫酸氢盐测序法分析myocardial DNA 为了探索全球甲基化状态，并专注于核糖体DNA（rDNA）甲基化， 核糖体生物发生与骨骼肌质量调节密切相关。在辅导阶段， 格兰特，我将学习必要的表观遗传技术，通过评估研究肌肉质量调节 肥大（PoWeR）和萎缩（后肢悬吊）期间的肌纤维表观遗传学。随着这些新 获得的技能，我将在独立的过程中从氧化和糖酵解肌肉中提取肌细胞DNA 阶段，以提供一个全面的肌肉老化表观遗传图谱。我也会在生命早期训练老鼠， 确定先前的肥大是否影响以后生活中对相同刺激的适应性，目的是 确定改善老年肌肉适应性的表观遗传线索。我的总体假设是 肥大将与肌肉类型特异性的全局和rDNA低甲基化相关，而卸载 并且衰老的特征在于肌肉类型特异性的超甲基化。我还假设早期生活 运动将与生命后期的rDNA低甲基化相关，这将与恢复 老年适应性肥大。该方案中的实验将为 开发基于表观遗传学的治疗方法，目的是随着衰老使骨骼肌年轻化。此外，委员会认为， 这个项目提供的培训机会将促进我作为一个独立的研究人员的成功， 在骨骼肌老化过程中肌肉质量调节的表观遗传学。

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.