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，并与慢性病状态下预后不良有关。确定萎缩的机制将使我们能够制定治疗策略来预防或治疗肌肉质量的损失。我们以前已经证明，在经历废用性或年龄相关性肌肉萎缩的肌肉中，会发生肌纤维核凋亡，并且在萎缩过程中，caspase非依赖的线粒体酶内切酶G(Endog)被移位到细胞核，可能导致细胞核中的DNA片段化。因此，我们假设萎缩的骨骼肌中发生的核凋亡是由endog介导的。在特定的目标1中，我们将使用endog基因敲除(KO)小鼠来确定在废用萎缩期间骨骼肌的凋亡反应是否需要endog。采用小鼠后肢悬吊(HS)模型，检测小鼠的核丢失、细胞凋亡和萎缩情况。此外，我们假设，随着氧化应激的增加和萎缩，核膜通透性的增加对于endog进入细胞核是必要的。野生型(WT)小鼠将被铁络合剂处理，并将测量内切酶移位、氧化损伤、核内切酶活性和核凋亡。在特定的目标2中，我们将研究细胞核变化对骨骼肌萎缩的影响。首先，将在体外研究WT小鼠(HS)后细胞核通透性的变化，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，我们还将通过激光消融消除肌核，并分析肌肉萎缩和蛋白质丢失，直接测试核的丧失是否会导致萎缩。最后，我们建议通过机械刺激撤除来建立废用性萎缩的体外模型，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，将测试endog在核凋亡中的作用。综上所述，拟议的实验将使我们能够确定内切酶介导的核凋亡在骨骼肌萎缩中的作用。这些结果不仅可以用来开发对抗肌肉萎缩的策略，还可以对其他组织中的细胞凋亡机制产生新的见解。与公共健康相关：肌肉萎缩是癌症、艾滋病和充血性心力衰竭等相关疾病，已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导致治疗干预，不仅在减少萎缩，而且在预防它。此外，识别肌肉中细胞核凋亡过程的潜在途径和分子也将增加我们对其他组织中的细胞凋亡的了解，可能导致在癌症等领域进行干预和治疗。 公共卫生相关性：骨骼肌萎缩是癌症、艾滋病和充血性心力衰竭等相关疾病，已被认为是老年人丧失独立性的主要原因。我们已经证明，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，不仅在减少萎缩方面，而且特别是在预防方面。此外，识别肌肉中细胞核凋亡过程的潜在途径和分子也将增加我们对其他组织中的细胞凋亡的了解，可能会导致在癌症等领域进行新的干预和治疗。

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.