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

摘要 人们仍然需要治疗方法来再生肌肉和/或防止肌肉丢失。目前的R21 该提议挑战了肌核无法复制以取代丢失的核或支持的教条 肥厚性生长。这一高风险、高回报的提议检验了肌核是Capa-C的整体假说。 因此，回答了一个长期悬而未决的问题。这一假说是由INTERI-INTERI-INTERI形成的。 Guing证据表明，在卫星细胞耗竭的肌肉中，肌核以及AP-2中的BrdU被掺入 在没有卫星细胞的情况下，亲本终生维持肌核。为我们的整体HY提供额外支持- 这一假说来自于开创性的研究，证明了哺乳动物心肌细胞分解蛋白质的能力。 分化并重新进入细胞周期，以及曾经被认为是有丝分裂后的其他细胞类型的发现 有复制的能力。为了检验整体假说，具体目的是测试肌核 在正常的笼子活动或超负荷诱导的肥大期间具有复制能力。建议数 这种方法在技术上是可行的，因为调查小组汇集了专业知识。该方法 使用一种新的转基因小鼠，允许在特定的时间段内对肌核进行GFP标记 在拟议的干预措施期间不会出现新的GFP标签的时间。在干预期间，小鼠 将通过饮用水给予氧化氢(D2O)，这将标记任何新合成的DNA 来自其他细胞来源的新的肌核将不含有GFP的一段时间。接下来的是- TION，GFP标记的肌核将用FACS分离，D2O的掺入用质谱仪测定。 在GFP+细胞中试试。考虑到这种设计中GFP标签的高度特异性，这种创新的方法允许 明确地确定是否有肌纤维核复制以及在什么条件下复制(S)。该项目具有很高的 重要的是因为支持这一假设的证据将从根本上改变该领域目前的不足- 骨骼肌基本生物学的立场。这样的证据将使肌核成为一种新的治疗焦油- 防止肌肉流失或促进肌肉生长。该项目具有创新性，因为它结合了一种新颖的 肌纤维特异性Tet-on小鼠和D2O标记以明确评估肌核DNA合成。如果 如果成功，这项拟议的研究将扭转长期以来的教条，并开创新的调查领域 和临床发展。未来的研究将表征肌原核周转的其他参数，如 以及机制研究，以确定肌核如何或何时复制。由此产生的影响是一条新的大道- 用于开发创新的治疗方法，以对抗随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.