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

总结 持续需要再生肌肉和/或防止肌肉损失的疗法。目前R21 一项提案挑战了肌核不能复制以取代丢失的细胞核或支持的教条， 肥大生长这个高风险，高回报的提议测试了肌核是capa的整体假设， 可复制，从而回答了一个长期悬而未决的问题。这一假设是由内- 有证据表明，在卫星细胞耗竭的肌肉中， 在没有卫星细胞的情况下，亲本终身维持肌核。为我们的整体健康提供更多支持- 假设来自于开创性的研究，证明了哺乳动物肌细胞的能力， 分化和重新进入细胞周期，以及发现其他细胞类型曾经被认为是有丝分裂后 有复制的能力。为了检验总体假设，特定目的旨在检验肌核 在正常的笼内活动期间或在过载诱导的肥大期间具有复制能力。拟议 由于调查小组汇集了专门知识，这种办法在技术上是可行的。的方法 使用了一种新的转基因小鼠，该小鼠允许在特定的时间段内特异性地对肌核进行GFP标记。 时间，使得在提议的干预期间不会发生新的GFP标记。在干预期间， 将通过饮用水给予氧化氘（D2 O），它标记任何新合成的DNA， 来自其他细胞来源的新肌核不含GFP的时间段。在干预之后， 在这一过程中，GFP标记的肌核将通过FACS分离，并且D2 O掺入通过质谱法测定。 尝试在GFP+细胞中。鉴于这种设计的GFP标记的高特异性，这种创新的方法允许 明确地确定是否有任何肌纤维核复制以及在何种条件下复制。该项目高度 重要的是，支持这一假设的证据将从根本上改变该领域目前的状况， 骨骼肌基础生物学的地位。这些证据将使肌核成为一种新的治疗方法， 防止肌肉萎缩或增加肌肉生长。该项目是创新的，因为它结合了一个小说 肌纤维特异性Tet-ON小鼠和D2 O标记以明确评估肌纤维DNA合成。如果 如果成功，这项研究将扭转长期存在的教条，并创造新的研究领域 临床发展。未来的研究将描述肌细胞核转换的其他参数， 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ave- 开发创新治疗方法以对抗随着年龄增长的肌肉损失和肌肉疾病 浪费

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.