The role of endonuclease G in nuclear apoptosis of atrophying skeletal muscle

核酸内切酶G在萎缩骨骼肌核凋亡中的作用

• 批准号: 7894182
• 负责人: Esther E Dupont-Versteegden
• 金额: $ 21.06万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894182
• 关键词: Ablation; Acquired Immunodeficiency Syndrome; Age; Animals; Apoptosis; Apoptotic; Atrophic; Attenuated; Biological Assay; Biological Models; Caspase; Cell Membrane Permeability; Cell Nucleus; Cessation of life; Chelating Agents; Chronic Disease; Congestive Heart Failure; DNA Fragmentation; Data; Deferoxamine; Development; Disease; Disuse Atrophy; Elderly; Enzymes; Event; Exhibits; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; GTP-Binding Proteins; Gastrocnemius Muscle; Goals; In Vitro; Intervention; Iron; Knock-out; Knockout Mice; Knowledge; Lasers; Lead; Limb structure; Location; Malignant Neoplasms; Measures; Mechanics; Mediating; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscle Proteins; Muscular Atrophy; NIH Program Announcements; Nuclear; Nuclear Envelope; Oxidative Stress; Pathway interactions; Permeability; Process; Protein Biosynthesis; Proteins; Public Health; Regulation; Research Project Grants; Research Proposals; Role; Skeletal Muscle; Stimulus; Structure; Study models; Suspension substance; Suspensions; System; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; United States National Institutes of Health; Wild Type Mouse; Withdrawal; Work; age related; anticancer research; base; combat; endonuclease; endonuclease G; in vitro Model; innovation; insight; interstitial cell; muscle form; novel; novel therapeutic intervention; outcome forecast; oxidative damage; prevent; public health relevance; research study; response; sarcopenia

DESCRIPTION (provided by applicant): Skeletal muscle atrophy is an important determinant in the loss of independence in elderly and is associated with poor prognosis in chronic disease states. Identifying mechanisms of atrophy will enable us to develop therapeutic strategies to prevent or treat the loss of muscle mass. We have shown previously that myofiber nuclear apoptosis occurs in muscles undergoing disuse-induced or age-associated muscle atrophy and that the caspase-independent mitochondrial enzyme endonuclease G (EndoG) is translocated to nuclei during atrophy, possibly inducing DNA fragmentation in a subset of nuclei. Therefore, we hypothesize that nuclear apoptosis which occurs in atrophying skeletal muscle is mediated by EndoG. In Specific Aim 1 we will determine whether EndoG is required for the apoptotic response in skeletal muscle during disuse atrophy using EndoG knockout (KO) mice. Mice will be hind limb suspended (HS) and nuclear loss, apoptosis and atrophy will be assayed. In addition, we hypothesize that increased permeability of the nuclear membrane due to elevated oxidative stress with atrophy is necessary for EndoG to enter the nucleus. Wild type (WT) mice will be treated with an iron-chelator and translocation of EndoG, oxidative damage, nuclear endonuclease activity and nuclear apoptosis will be measured. In Specific Aim 2 we will study the consequences of nuclear changes on skeletal muscle atrophy. First, permeability changes of nuclei of WT mice after (HS) will be studied in vitro and correlated to oxidative stress to determine whether this is directly responsible for the entry of molecules into the nucleus. Additionally, we will test directly whether the loss of nuclei induces atrophy by eliminating muscle nuclei by laser ablation and assaying for muscle atrophy and protein loss. Lastly, we propose to develop an in vitro model of disuse atrophy by mechanical stimulus withdrawal and compare this to an in vitro atrophy model of oxidative stress. The role of EndoG in nuclear apoptosis in these models will be tested. In summary, the proposed experiments will enable us to determine the role of EndoG-mediated nuclear apoptosis in skeletal muscle atrophy. These results may be not only be used to develop strategies to combat muscle atrophy, but may also yield novel insight into mechanisms of apoptosis in other tissues. Relevance to public health: Muscle atrophy is associated diseases such as cancer, AIDS, and congestive heart failure, and has been suggested as a main contributor to the loss of independence in elderly. We showed that apoptosis is an important event in the process of muscle atrophy. Identifying underlying mechanisms may lead to therapeutic interventions, not just in decreasing atrophy, but in preventing it. Additionally, identifying pathways and molecules underlying the process of nuclear apoptosis in muscle will increase our knowledge about apoptosis in other tissues as well, likely leading to interventions and treatments in fields such as cancer. PUBLIC HEALTH RELEVANCE: Skeletal muscle atrophy is associated diseases such as cancer, AIDS, and congestive heart failure, and has been suggested as a main contributor to the loss of independence in elderly. We have shown that nuclear apoptosis is an important event in the process of muscle atrophy. Identifying underlying mechanisms may lead to new therapeutic interventions, not just in decreasing atrophy, but particularly in preventing it. Additionally, identifying pathways and molecules underlying the process of nuclear apoptosis in muscle will increase our knowledge about apoptosis in other tissues as well, possibly leading to new interventions and treatments in fields such as cancer.

描述（由申请人提供）：骨骼肌萎缩是老年人独立性丧失的重要决定因素，并且与慢性疾病状态下的不良预后相关。确定萎缩的机制将使我们能够制定预防或治疗肌肉质量损失的治疗策略。我们之前的研究表明，肌纤维核凋亡发生在废用性或年龄相关性肌肉萎缩的肌肉中，并且在萎缩过程中，不依赖于caspase的线粒体酶内切酶G （EndoG）易位到核中，可能导致一部分核中的DNA断裂。因此，我们假设萎缩骨骼肌的核凋亡是由EndoG介导的。在Specific Aim 1中，我们将使用EndoG基因敲除（KO）小鼠，确定EndoG是否需要骨骼肌在废用性萎缩期间的凋亡反应。小鼠后肢悬吊（HS），检测细胞核丢失、细胞凋亡和萎缩。此外，我们假设由于氧化应激升高和萎缩导致核膜通透性增加是EndoG进入细胞核的必要条件。野生型（WT）小鼠用铁螯合剂和EndoG易位处理，测量氧化损伤、核内切酶活性和核凋亡。在具体目标2中，我们将研究核变化对骨骼肌萎缩的影响。首先，我们将在体外研究（HS）后WT小鼠细胞核的通透性变化，并研究其与氧化应激的相关性，以确定这是否直接导致分子进入细胞核。此外，我们将通过激光消融消除肌肉核并测定肌肉萎缩和蛋白质损失来直接测试核的丢失是否会导致萎缩。最后，我们建议建立机械刺激退出的废用性萎缩体外模型，并将其与氧化应激的体外萎缩模型进行比较。在这些模型中，我们将测试EndoG在核凋亡中的作用。总之，所提出的实验将使我们能够确定内啡肽介导的核凋亡在骨骼肌萎缩中的作用。这些结果可能不仅用于制定对抗肌肉萎缩的策略，而且可能对其他组织的细胞凋亡机制产生新的见解。与公共卫生的相关性：肌肉萎缩与癌症、艾滋病和充血性心力衰竭等疾病有关，并被认为是老年人丧失独立性的主要原因。我们发现细胞凋亡是肌肉萎缩过程中的一个重要事件。确定潜在的机制可能导致治疗干预，不仅在减少萎缩，而且在预防它。此外，确定肌肉核细胞凋亡过程的途径和分子也将增加我们对其他组织细胞凋亡的了解，可能导致癌症等领域的干预和治疗。