In skeletal muscle (SKM), gene expression of transcription factors regulating myogenesis are dependent on thyroid hormone (TH) signal transduction. Expression of myogenic regulatory factors may be altered due to dysregulated TH metabolism, which may result in SKM dysfunction and intolerance to exercise in individuals with hypothyroidism. PURPOSE: To use an in vitro model of hypothyroidism to test the hypothesis that SKM cells will have dysregulation in transcription factors regulating myogenesis. Additionally, the exercise mimetic, formoterol, was used to determine the effects of exercise signaling during myogenesis. METHODS: Human SKM myoblasts (n = 6 per group) were cultured and differentiated until mature myotube formation (Day 6). Groups included control cells (CON), TH depleted cells (ThD), and TH depleted cells plus formoterol stimulation (ThD+F; 30nM for 3h). Total RNA was extracted during mid-myogenesis (Day 4) and at terminal differentiation (Day 6). Gene expression for myogenic regulatory factors (Myf5, MyoD, MyoG) was determined by qPCR. Data were analyzed by repeated measures ANOVA. RESULTS: Significant differences between conditions and time points are detailed in Table 1. CONCLUSION: TH depletion had no effect on MyoG but did reduce the expression of both Myf5 and MyoD at both D4 and D6. Additionally, ThD+F resulted in the lowest expression of MyoG and MyoD for both time points. These results indicate TH depletion and formoterol stimulation may inhibit myotube maturation. This work was supported by a Texas ACSM SRDA grant.Table 1