Semester

Summer

Date of Graduation

2014

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Exercise Physiology

Committee Chair

Stephen E. Alway

Committee Co-Chair

Randall W. Bryner

Committee Member

Junaith S. Mohamed

Committee Member

Emidio Pistilli.

Abstract

Satellite cells are normally a quiescent skeletal muscle stem cell pool that upon activation rapidly proliferate and differentiate to repair damaged muscle fibers. The paired box transcription factor 7 (Pax7) is the canonical biomarker for quiescent, activated, and proliferating satellite cells and is rapidly down-regulated upon differentiation into myofibers. However, the factors that regulate the modulation of Pax7 are largely unknown. Post-translational modifications to Pax7 provide a viable means to activate or deactivate this transcription factor through the signaling cascade of skeletal muscle repair and regeneration. The sirtuin 1 (Sirt1) protein, a histone deacetylase, has been shown to be involved in many cellular processes, including the differentiation of myoblasts. Although Sirt1 is noted for its role in the differentiation of myoblasts, it is not known if it directly interacts with Pax7. The general control of amino acid synthesis protein 5-like 2 (GCN5), an acetyltransferase, is known to work in conjunction with Sirt1 modulating PGC-1&agr; in hepatic gluconeogenesis. With this information, it is logical to test if GCN5 also works with Sirt1 in the satellite cell response to muscle injury. The primary objective of this study is to determine the role of GCN5 and Sirt1 on Pax7-mediated regulation of satellite cell function. We tested the novel hypothesis that GCN5 is a binding partner with Pax7 and acts on Pax7 via acetylation leading to satellite cell activation and proliferation while Sirt1 is another binding partner of Pax7 and acts on Pax7 via deacetylation. The tibialis anterior (TA) muscles of wild-type mice were injected with cardiotoxin (CTX) to induce muscle injury. The TA muscles where harvested 4, 7, 14, or 21 days post-injection to analyze the satellite cell response to regulate muscle repair following the CTX injury. Western Blot data showed that the total protein abundance of Pax7, GCN5, and acetylated protein at 57kD (Pax7 molecular weight) were significantly up-regulated 4, 7, and 14 days post CTX injection, but had returned to basal levels by 21 days post CTX injection. From 4 through 14 days post injection Pax7 abundance was ∼592%, 204%, and 219% greater compared to control muscles. GCN5 abundance was ∼215%, 330%, and 213% greater from 4 through 14 days post injection, and acetylated protein at 57kD was ∼651%, 291%, and 404% greater compared to controls. The abundance of Sirt1 protein was significantly increased at 14 days post CTX injection with ∼ 259% greater abundance compared to control muscles and approached basal levels by 21 days post CTX injection. Although not significant, there was ∼111% greater abundance of Sirt1 protein 7 days post CTX injection relative to control muscles. Immunoprecipitation studies showed that GCN5 was directly associated with Pax7 during all recovery time points, with 21 days post CTX injury not having as strong association compared to the other time points. The membrane was then stripped and probed for acetylated lysine, which mirrored the GCN5 association pattern. Sirt1 was also tested for interaction and showed to be directly interacting with Pax7 during the 7, 14, and 21 days post CTX injury time points. Immunohistochemistry confirmed that Pax7 and GCN5 were co-localized to muscle nuclei in the 4 day regenerating muscles; this confirms that Pax7 and GCN5 interact during activation and proliferation of satellite cells. Immunohistochemistry also confirmed that Pax7 and Sirt1 were co-localized in the 14 day regenerating muscles; this confirms that Pax7 and Sirt1 interact during the differentiation of myoblasts. These novel data support the hypotheses that GCN5 acetylates Pax7 to rapidly activate and proliferate satellite cells in response to skeletal muscle damage while Sirt1 deacetylates Pax7 to differentiate myoblasts.

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