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.

概括持续需要再生肌肉和/或防止肌肉损失的疗法。目前的R21 该提案挑战了肌核无法复制以取代丢失的细胞核或支持物的教条肥大性生长。这个高风险、高回报的提议测试了肌核具有能力的总体假设。复制能力，从而回答了一个长期悬而未决的问题。这个假说是由内部形成的有力的证据表明，在卫星细胞耗尽的肌肉以及 AP 中，BrdU 被肌核掺入在没有卫星细胞的情况下，母体终生维持肌核。对我们整体发展的额外支持该假设来自开创性研究，证明哺乳动物肌细胞有能力去分化并重新进入细胞周期，以及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，具体目标是检验肌核是否具有在正常的笼子活动期间或在过载引起的肥大期间复制的能力。拟议的由于调查团队汇集了专业知识，该方法在技术上是可行的。方法使用一种新型转基因小鼠，可以在指定的时间内对肌核进行 GFP 标记时间，以便在建议的干预期间不会出现新的 GFP 标记。干预期间小鼠将通过饮用水注入氧化氘 (D2O)，在过程中标记任何新合成的 DNA 来自其他细胞来源的新肌核不含有 GFP 的一段时间。干预后化，GFP标记的肌核将通过FACS分离，并通过质谱测定D2O掺入- 在 GFP+ 细胞中尝试。鉴于这种设计的 GFP 标记具有高度特异性，这种创新方法可以明确确定是否有任何肌纤维核复制以及在什么条件下复制。该项目具有高度意义重大，因为支持该假设的证据将从根本上改变该领域当前的不足骨骼肌的基本生物学地位。这些证据将使肌核成为一种新的治疗目标防止肌肉损失或增加肌肉生长。该项目具有创新性，因为它结合了新颖的肌纤维特异性 Tet-ON 小鼠和 D2O 标记可明确评估肌核 DNA 合成。如果如果成功的话，拟议的研究将扭转长期存在的教条并创造新的研究领域和临床开发。未来的研究将描述肌核周转的其他参数，如以及确定肌核如何或何时复制的机制研究。由此产生的影响是一个新的途径开发创新疗法以对抗随年龄增长和肌肉疾病而导致的肌肉损失的必要性浪费。