Enhanced Myonuclear Alertness with Prolonged Satellite Cell Depletion

The maintenance of skeletal muscle mass and function is of clinical importance since its decline is associated with increased morbidity and mortality as well as a marked deterioration in the quality of life. Increases in skeletal muscle size occur primarily through hypertrophic growth of myofibers. This hypertrophic growth is accompanied by an increase in the number of nuclei per myofiber. The increase in myonuclei is thought to be necessary for muscle hypertrophy. Given that myonuclei are post-mitotic, the accretion of “new” myonuclei during hypertrophy is the result of satellite cell fusion; satellite cells are the primary stem cell population found in adult skeletal muscle. In contrast to post-natal development, skeletal muscle of mature (5 months of age) mice are capable of hypertrophic growth independent of satellite cell fusion; however, like post-natal development, skeletal muscle of juvenile (2 months of age) mice are unable to hypertrophy when myonuclear accretion is prevented as the result of satellite cell depletion. I have acquired preliminary data showing skeletal muscle hypertrophy was enhanced in satellite cell-depleted, juvenile mice which were allowed to age to maturity. Based on this exciting finding, I hypothesize that during the 3-month maturation period, resident myonuclei, of satellite cell depleted muscle, undergo epigenetic remodeling that facilitates ribosome biogenesis thereby promoting enhanced hypertrophic growth of skeletal muscle. To test this hypothesis, a novel genetic mouse model will be used, designated the Quad1 (Q1) mouse, which will allow simultaneous GFP labeling of myonuclei and the depletion of satellite cells within the same mouse. The Q1 mouse will be used to test the hypothesis by pursuing the following specific aim. I will use fluorescence-activated cell sorting to purify GFP-labeled myonuclei for deep RNA-seq and reduced bisulfite sequencing analyses to identify differentially expressed or demethylated genes involved with ribosome biogenesis in skeletal muscle of satellite cell-depleted, juvenile-matured Q1 mice in response to a hypertrophic stimulus. Furthermore, I will use immunohistochemistry to determine if satellite cell-depleted, juvenile-matured Q1 mice show enhanced hypertrophy as a result of greater ribosome biogenesis. The findings from the proposed experiments are expected to provide evidence for a novel mechanism used by resident myonuclei to compensate for the loss of satellites which promotes enhanced muscle hypertrophy as the result of greater ribosome biogenesis. The proposed experiments will provide the applicant with outstanding training in murine husbandry, the use of inducible transgenic mouse models, murine surgical techniques, FACS, RNA-seq, RRBS, and extensive computational bioinformatics as well as mentorship in grantsmanship, networking and lab management.