DNA turnover in myofibers is an unrecognized mechanism for maintaining skeletal muscle health

肌纤维中的 DNA 更新是维持骨骼肌健康的一种未被认识的机制

基本信息

批准号：
10065144
负责人：
Benjamin Francis Miller
金额：
$ 24.17万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-15 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10065144
关键词：
Ablation Age Aging Area Belief Biology Birds Bromodeoxyuridine Cardiac Myocytes Cell Cycle Cell Cycle Arrest Cell Nucleus Cell fusion Cells Cellular Stress Clinical DNA DNA biosynthesis Data Deuterium Oxide Development Disease Event Exercise Exploratory/Developmental Grant Fluorescence-Activated Cell Sorting Frequencies Future G0 Phase Goals Growth Health Hepatocyte Human Hypertrophy Intervention Investigation Label Lead Maintenance Mass Spectrum Analysis Mechanics Metabolic Methods Mitotic Mus Muscle Muscle Cells Muscle Fibers Muscular Atrophy Myopathy Natural regeneration Paper Parents Process Regulation Reporting Research Seminal Signal Transduction Skeletal Muscle Source Specificity Sum Testing Tetanus Helper Peptide Therapeutic Time Transgenic Mice Work base cell growth cell type clinical development combat design drinking water high reward high risk improved in vivo innovation mouse model muscle form muscle hypertrophy new therapeutic target novel novel strategies preservation prevent regenerative satellite cell skeletal therapeutic target

项目摘要

SUMMARY There is a continued need for therapies to regenerate muscle and/or prevent muscle loss. The current R21 proposal challenges the dogma that myonuclei are unable to replicate in order to replace lost nuclei or support hypertrophic growth. This high-risk, high-reward proposal tests the overall hypothesis that myonuclei are capa- ble of replication, thus answering a long-standing unresolved question. This hypothesis was formed by intri- guing evidence showing BrdU incorporation by myonuclei in satellite cell-depleted muscle as well as the ap- parent lifelong maintenance of myonuclei in the absence of satellite cells. Additional support for our overall hy- pothesis comes from pioneering studies demonstrating the capability of mammalian myocytes to de- differentiate and re-enter the cell cycle, and the discovery that other cell types once thought to be post-mitotic have the ability to replicate. To test the overall hypothesis, the specific aim is designed to test if myonuclei have the ability to replicate during regular cage activity or during overload-induced hypertrophy. The proposed approach is technically feasible because of the assembled expertise of the investigative team. The approach uses a novel transgenic mouse that allows for GFP-labeling of myonuclei specifically during a defined period of time such that no new GFP labeling will occur during the proposed interventions. During the intervention mice will be administered deuterium oxide (D2O) via drinking water, which labels any newly synthesized DNA during a period of time when new myonuclei from other cellular sources will not contain GFP. Following the interven- tion, GFP-labeled myonuclei will be isolated by FACS, and D2O incorporation determined by mass spectrome- try in GFP+ cells. Given the high specificity of GFP labeling with this design, this innovative approach allows for unambiguously determining if any myofiber nuclei replicated and under what condition(s). The project is highly significant because evidence supporting the hypothesis would radically transform the field's current under- standing of the basic biology of skeletal muscle. Such evidence would make myonuclei a novel therapeutic tar- get to prevent muscle loss or increase muscle growth. The project is innovative because it combines a novel myofiber-specific Tet-ON mouse and D2O labeling to unambiguously assess myonuclear DNA synthesis. If successful, the proposed research would reverse a long-standing dogma and create new areas of investigation and clinical development. Future studies would characterize additional parameters of myoncuclei turnover, as well as mechanistic studies to determine how or when myonuclei replicate. The resulting impact is a new ave- nue for the development of innovative treatments to combat muscle loss with age and diseases of muscle wasting.

概括持续需要疗法来再生肌肉和/或预防肌肉损失。当前的R21 提案挑战肌核无法复制以取代丢失的核或支持的教条肥厚的生长。这种高风险的高回报提案检验了整体假设，即肌核是capa- 复制，从而回答了一个长期以来的尚未解决的问题。该假设是由表明肌核中肌核纳入卫星细胞耗尽的肌肉以及AP-的Guing证据在没有卫星细胞的情况下，母体终身维持肌核。对我们整体的额外支持 pothesis来自开创性的研究，证明了哺乳动物肌细胞的能力区分和重新输入细胞周期，并发现其他细胞类型曾经被认为是有丝分裂后的具有复制能力。为了检验总体假设，具体目的旨在测试肌核是否是否具有在常规笼子活性或过载引起的肥大期间复制的能力。提议由于调查团队的组装专业知识，方法在技术上是可行的。方法使用一种新型的转基因小鼠，该小鼠允许在定义的时期内专门针对肌核的GFP标记时间使得在拟议的干预措施期间不会发生新的GFP标签。干预小鼠期间将通过饮用水施用氧化氘（D2O），该饮用水标记任何新合成的DNA 来自其他细胞源的新肌核不会包含GFP的时间。介入 - Tion，GFP标记的肌核将通过FACS分离，D2O掺入由质谱 - 尝试使用GFP+细胞。考虑到GFP标签具有这种设计的高特异性，这种创新的方法允许明确确定是否复制任何肌纤维核并在什么情况下复制。该项目高度重要的是因为支持该假设的证据将从根本上改变该田地的电流 - 骨骼肌的基本生物学的站立。这样的证据会使肌核素成为一种新颖的治疗性焦油预防肌肉损失或增加肌肉生长。该项目具有创新性，因为它结合了一本小说肌纤维特异性的小鼠和D2O标记，以明确评估肌核DNA合成。如果成功的研究将扭转长期的教条，并创建新的调查领域和临床发展。未来的研究将表征肌酸周转的其他参数，例如以及确定肌核如何或何时复制的机械研究。由此产生的影响是一个新的AVE- 为开发创新治疗以与年龄和肌肉疾病打击肌肉丧失的创新治疗方法浪费。