Date of Award

9-1-2011

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Kinesiology

First Advisor

Priscilla M. Clakson

Second Advisor

Lawrence M. Schwartz

Third Advisor

Edward P. Debold

Subject Categories

Kinesiology

Abstract

Skeletal muscle is dynamic and actively regenerates following damage or altered functional demand. Regeneration is essential for the maintenance of muscle mass and, when dysregulated as a result of disease or aging, can lead to losses in functional capacity and increased mortality. Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. Therefore, the overall objective of this dissertation was to characterize early molecular alterations in human skeletal muscle to strenuous exercise known to induce a muscle regenerative response. Thirty-five subjects completed 100 eccentric (muscle lengthening) contractions (EC) of the knee extensors with one leg and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. A well-powered transcriptomic screen and network analysis using Ingenuity Pathway software was first conducted on mRNA from the biopsy samples. Network analysis identified the transcription factor NF-kappaB (NF-kB) as a key molecular element affected by EC. Conformational qRT-PCR confirmed alterations in genes associated with NF-kappaB. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples showed a 1.6 fold increase in NF-kB DNA binding activity following EC. Immunohistochemical experiments then localized the majority of NF-kB positive nuclei to cells in the interstitium, which stained positive for markers of pericyte cells and not satellite cells. To ascertain the mechanistic significance of NF-kB activation following muscle damage, in vitro analyses were carried out using a novel primary pericyte/myoblast co-culture model. Primary pericyte/myoblast co-culture experiments demonstrated that pericytes, transfected with a DNA vector designed to drive NF-kB activation, enhanced proliferation and inhibited myogenic differentiation of co-cultured skeletal muscle myoblasts. Furthermore, reduced NF-kB activation led to enhanced myogenic potential of primary pericytes. Taken together, the data in this dissertation suggest that NF-kB dependent signaling in pericytes regulates myogenic differentiation in a cell- and non-cell autonomous manner and may affect the early regenerative response following muscle damage by inhibiting differentition and promoting proliferation of muscle satellite cells.

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