Date of Graduation

5-2022

Document Type

Thesis

Degree Name

Master of Science in Kinesiology (MS)

Degree Level

Graduate

Department

Health, Human Performance and Recreation

Advisor/Mentor

Tyrone A. Washington

Committee Member

Nicholas P. Greene

Second Committee Member

Kevin A. Murach

Third Committee Member

Michelle Gray

Keywords

cell culture, growth differentiation factor 5, obesity, sarcopenia, sarcopenic obesity

Abstract

Sarcopenic obesity attributes to skeletal muscle loss more than sarcopenia and obesity alone. Individuals with SO suffer from the comorbidity of excess body fat and concurrent muscle mass loss due to aging. Growth differentiation factors (Gdfs) have never been recognized as playing a role in skeletal muscle maintenance in those with SO. Specifically, Gdf5, has been recognized as playing a part in Bone Morphogenic Protein signaling to activate protein synthesis and deactivate protein degradation via SMAD 1/5/8 and SMAD 4 complex. Using RNA sequencing, Gdf5 was identified as being significantly upregulated in SO mice. Purpose: To determine the cellular role that Gdf5 plays in sarcopenic obese skeletal muscle. Methods: In vitro experimentation was conducted by using C2C12 cell culture. Cells were grown to 70-80% confluency with 10% growth media and differentiated out for 6 days in differentiation media. Lipofectamine transfection was used to transfect myoblasts with an empty vector control plasmid or a shRNA for Gdf5 knockdown. Western blot was used to measure Gdf5 protein expression. Myotube diameter were measured for each group. Markers for myogenic regulation (Cyclin D1, MyoD, MyoG), protein degradation (Atrogin, MuRF1) and inflammatory markers (IL-6, TNF-α) were assessed using RT-PCR for mRNA abundance. Results: There was a 62% and 30% (p<0.05) decrease of Gdf5 protein expression at 28 kDa and 55 kDa in the shRNA 1 group. The shRNA 2 and 3 groups had decreases of 36% and 27% (p<0.05) at 55 kDa. respectively. Myotube size between groups were unchanged. Gdf5 and Dnmt3a mRNA abundance increased in the Gdf5 shRNA group when compared to control group by 51% and 14% (p<0.05) respectively. In the Gdf5 shRNA group, mRNA abundance of MyoD was decreased by 17% and MyoG was increased by 51% (p<0.05). mRNA abundance of protein degradation marker, MuRF-1, was increased by 39.2% (p<0.05) in the Gdf5 shRNA group.

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