Date of Completion

Spring 5-1-2015

Thesis Advisor(s)

Dr. Sarah Reed

Honors Major

Animal Science

Abstract

During times of stress, systemic pro-inflammatory cytokine levels are increased, which may prevent optimal growth and development of muscle and/or induce muscle atrophy. Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine involved in stress and disease responses, but little is known about how IL-1β affects myoblast function. We hypothesized that IL-1β would decrease myoblast proliferation and/or differentiation. To test this hypothesis, C2C12 mouse myoblasts were treated with 0.1 ng/mL or 1.0 ng/mL of IL-1β, or carrier only (control). To determine proliferation rate, myoblasts were plated at 2.6x103 cells/cm2 and cultured for 48 h in the presence or absence of IL-1β. Cells were pulsed with bromodeoxyuridine (BrdU), fixed, and immunostained. The number of BrdU positive cells was quantified as a percent of total nuclei (identified by Hoescht 33342). To determine if IL-1β affected fusion, myoblasts were plated at 2.0x104 cells/cm2 in growth media for 48 h at which time media was changed into differentiation media supplemented with 0.1 ng/mL or 1.0 ng/mL of IL-1β, or carrier only. Cells were immunostained with myosin heavy chain (MyHC) and Hoescht 33342. Fusion index was determined by quantifying the number of nuclei within multinucleated myotubes divided by total nuclei. Finally, to determine the effect of IL-1β on myotube size, myoblasts were cultured for 48 h in growth media and 48 h in differentiation media. Cells were cultured for an additional 48 h in the presence or absence of IL-1β, fixed and immunostained for MyHC. Myotube diameter and fusion index were quantified. All data was analyzed using ANOVA in GraphPad Prism followed by Tukey’s test for multiple comparisons. There were no significant effects of IL-1β on proliferation or fiber diameter (P ≥ 0.05). However, fusion was decreased 13.5% in myoblasts treated with 1.0 ng/mL of IL-1β (P ≤ 0.05). In conclusion, IL-1β decreases myoblast fusion, but does not affect proliferation or fiber diameter. These results suggest that IL-1β may contribute to poor muscle growth by decreasing fusion of myoblasts into existing myofibers, preventing optimal hypertrophy.

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