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

描述（由申请人提供）：骨骼肌萎缩是老年人丧失独立性的重要决定因素，与慢性疾病状态的预后不良相关。确定萎缩的机制将使我们能够制定治疗策略，以预防或治疗肌肉质量的损失。我们以前已经表明，肌纤维核凋亡发生在肌肉经历废用诱导或年龄相关的肌肉萎缩和半胱天冬酶非依赖性线粒体内切酶G（EndoG）易位到细胞核在萎缩过程中，可能会诱导DNA片段化的一个子集的核。因此，我们推测在萎缩的骨骼肌中发生的核凋亡是由EndoG介导的。在具体目标1中，我们将使用EndoG敲除（KO）小鼠确定在废用性萎缩期间骨骼肌中的凋亡反应是否需要EndoG。将小鼠后肢悬吊（HS），并测定细胞核丢失、细胞凋亡和萎缩。此外，我们假设，由于氧化应激升高伴萎缩导致核膜通透性增加是EndoG进入细胞核所必需的。将用铁螯合剂处理野生型（WT）小鼠，并测量EndoG的易位、氧化损伤、核内切核酸酶活性和核凋亡。在具体目标2中，我们将研究骨骼肌萎缩的核变化的后果。首先，将在体外研究（HS）后WT小鼠细胞核的渗透性变化，并将其与氧化应激相关联，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细胞核并测定肌肉萎缩和蛋白质损失来直接测试细胞核损失是否诱导萎缩。最后，我们建议开发一个体外模型的废用性萎缩的机械刺激撤退，并比较这一体外萎缩模型的氧化应激。将测试EndoG在这些模型中的核凋亡中的作用。总之，所提出的实验将使我们能够确定EndoG介导的细胞核凋亡在骨骼肌萎缩中的作用。这些结果不仅可以用于开发对抗肌肉萎缩的策略，而且还可以对其他组织中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。识别潜在的机制可能会导致治疗干预，不仅在减少萎缩，但在防止它。此外，识别肌肉细胞核凋亡过程中的途径和分子将增加我们对其他组织细胞凋亡的知识，以及，可能导致在癌症等领域的干预和治疗。 公共卫生相关性：骨骼肌萎缩与癌症、艾滋病、充血性心力衰竭等疾病有关，是老年人丧失独立能力的主要原因。我们已经表明，细胞核凋亡是肌肉萎缩过程中的一个重要事件。识别潜在的机制可能会导致新的治疗干预措施，不仅在减少萎缩，但特别是在防止它。此外，识别肌肉细胞核凋亡过程中的途径和分子将增加我们对其他组织细胞凋亡的知识，以及，可能导致新的干预措施和治疗领域，如癌症。

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.