The Role of Endonuclease G in nuclear apoptosis of atrophying skeletal muscle

Abstract 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 Endoc is required for the apoptotic response in skeletal muscle during disuse atrophy using EndoG knockout (KO) mice. Mice will be hind limb suspended (hIS) 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 (WI) 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 WI 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.