In vivo study of satellite cell addition and function in hypertrophying skeletal muscle

Skeletal muscle fibers contain many nuclei. It is believed that each nucleus control a certain amount of cytoplasm, called the myonuclear domain, and that a constant myonuclear domain is maintained during changes in fiber size. This implies that in order for fibers to increase in size, new nuclei are added through proliferation and fusion of muscle stem cells called satellite cells. Many studies have reported that the number of myonuclei increase during muscle fiber hypertrophy and it is believed that addition of myonuclei are necessary for the muscle fiber to increase in size. However, a recent finding that muscle hypertrophy occurs in a transgenic model were satellite cells are ablated, questions the idea that satellite cells are necessary for fiber hypertrophy. In my project I have used a transgenic mouse model where satellite cells can be ablated in adult animals. Contradictory to a previous report using this model, synergist ablation did not lead to any hypertrophy in mouse lacking satellite cells in our study. Our findings strongly suggest that satellite cells and insertion of new myonuclei are necessary for muscle hypertrophy. Satellite cells are responsible for both muscle growth and regeneration of our muscles. To further study the necessity of satellite cells I visited Tom A. Rando lab at Stanford University. In this lab I studied the addition of satellite cells using transgenic models expressing either different fluorescent reporters or luciferase. Furthermore, I studied the effect of exercise on satellite cells in old animals. For these experiments satellite cells were sorted by FACS, and we studied the effect on muscle plasticity, and also molecular studies of the satellite cells. I also developed a method to sort in vivo-fixed satellite cells by FACS. Our experiments give new knowledge of the regulation of satellite cell addition and function in skeletal muscle. We have shown that satellite cell addition is a prerequisite for muscle hypertrophy. Furthermore we have characterized the satellite cell-intrinsic effects of exercise. This insight will allow for the design of therapeutic strategies that augment loss of muscle mass due to chronic disease and aging, and development of pharmacologic strategies that replicate or even surpass the effect of exercise. Muscle wasting and satellite cell dysfunction also occurs in a numerous diseases including muscular dystrophy, cancer, obesity, diabetes, and chronic pulmonary disease. Elucidation of function of satellite cell addition for adult muscle hypertrophy could also point to therapeutic strategies for maintaining muscle repair ability in these diseases.

Skeletal muscle fibres are multinucleated. It has been believed that each nucleus serve a certain amount of cytoplasm, and that a constant myonuclear domain is maintained during changes in fibre size. This implies that in order for fibres to increase in size, new nuclei are added through proliferation and fusion of satellite cells, and that atrophy is accompanied by loss of nuclei by apoptosis within intact fibres. Several studies investigating skeletal muscle atrophy have reported that the myonuclear number are reduced during atrophy. I have worked in the lab of Kristian Gundersen in Oslo where in vivo imaging techniques can be used to follow single myonuclei with time-lapse microscopy. Such techniques have revealed that myonuclei are not lost from muscle fibres during atrophy. But, I have participated in confirming that myonuclear number increase during hypertrophy and that increased number of myonuclei facilitate muscle hypertrophy. However, recent findings that muscle hypertrophy occurs in transgenic models were satellite cells are ablated, questions the idea that myonuclei are necessary for fiber hypertrophy. The effect of satellite cell ablation during hypertrophy were only investigated in short term studies and it is not known if such muscles remain fully functional over time or if the lack of nuclei from satellite cells is compensated for. I plan to study hypertrophy in transgenic mice where the satellite cells have been ablated, and to use advanced cloning techniques to develop a new transgenic mouse where satellite cells are visualized in vivo. By visiting foreign groups I will investigate the properties of hypertrophic muscles with few myonuclei by measuring muscle force in single fibres, protein turnover and protein quality. I also hypothesize that myonuclear mitosis or aneploidy might play a compensatory role in absence of satellite cells, and I will investigated this possibility with fluorescence in situ hybridization (FISH).