Investigating the Relationship Between the Molecular Circadian Clock and Muscle Satellite Cell Fate

Skeletal muscle is a dynamic tissue capable of rapid regeneration. The functional unit of skeletal muscle, known as myofibers, contains terminally differentiated, post-mytotic myonuclei. These myofibers are often damage as a result of injury, exercise, natural turnover, or disease. Upon trauma, a rare population of muscle stem cells, known as satellite cells, mount a coordinated regenerative response to repair the damage. The regenerative response is well orchestrated and precisely coordinated in time such that the myofiber that exists after regeneration is identical to the myofiber that existed before trauma. The coordination of the individual events within the regenerative response is currently uncharacterized, but is potentially facilitated by the molecular circadian clock. The molecular clock is a tightly regulated feedback loop that oscillates over a 24 hour period and functions to coordinated various cellular functions. I am interested in understanding if the molecular clock coordinates the timing of the regenerative response by examining differentiation and quiescence. Using qRT-PCR, I examined the mRNA levels of three key genes expressed at different points throughout regeneration including the satellite cell specific gene Pax 7 and the differentiation markers myogenin and Myf6. In a myoblast cell line and in primary cells treated with chemical circadian inhibitors, Pax7 was unregulated whereas myogenin and My6 were down regulated. Using immunofluorescence, I found that myogenin was expressed less when the cells were treated with circadian inhibitors. I also used immunofluorescence to determine that circadian inhibition increased the number of undifferentiated cells that were not actively in the cell cycle. Overall, phenotypes of cells treated with circadian inhibitors are more undifferentiated than cells with a functioning clock, suggesting the clock plays a critical role in managing and coordinating regeneration in skeletal muscles.