Date of Graduation

2017

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Stephen E Alway

Committee Co-Chair

Randall W Bryner

Committee Member

Junaith S Mohamed

Committee Member

Emidio E Pistilli

Abstract

The postnatal growth and regenerative capacity of skeletal muscle rely on the successful activation and differentiation of muscle stem cells known as satellite cells. Although the NAD+-dependent protein deacetylase sirtuin-2 (Sirt2) has been suggested to have a role as a redox sensor during myogenesis, and in the regulation of metabolism, microtubule dynamics, and exit from the cell cycle exit, it is not known if Sirt2 has a regulatory role in muscle stem cell differentiation. This study aimed to elucidate the regulation of Sirt2 during the myogenic program of muscle satellite cells. The C2C12 mouse myoblast cell line was used to examine the regulatory mechanisms of Sirt2 during the differentiation process. Several cell lines were used during experimentation including wild type (-) cells, cells supplemented with the Sirt2 inhibitor BML-266, and cells which were transfected with CRISPR/Cas9 plasmids to generate both Sirt2 knockdown (SKO) and control plasmid (CC) lines. Assays were conducted at time points from 0h-96h following the induction of differentiation to assess Sirt2 regulation and downstream interactions. Sirt2 mRNA and protein expression were elevated during the early stages of differentiation at the 12h time point in the (-) cells. A significant decrease in the myotube fusion index of both BML-266 and SKO cell lines was observed, indicating Sirt2 deacetylase activity is important in regulating the normal formation of myotubes. Immunoprecipitation experimentation was performed to evaluate Sirt2 protein interactions and possible downstream signaling targets. Sirt2 was observed to interact with the cell cycle inhibitor retinoblastoma (pRB), as well as p21 and E2F1 a regulator of cellular proliferation. The acetylation status of E2F1 was significantly decreased at the 12h time point coinciding with the increased Sirt2 protein expression observed at the same point. These results indicate that Sirt2 mediates its control of myogenesis through interaction and complex formation with proteins associated with the cell cycle, and that this interaction mediates exit from the cell cycle and the induction of terminal differentiation leading to the formation of myofibers. Elucidation of Sirt2 as a regulator of differentiation during myogenesis thus presents a possible therapeutic target in conditions such as aging, as expression of the sirtuins and their cofactor NAD+ both decrease in aged phenotypes.

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