Abstract

Skeletal muscle growth is important for organism development. It also occurs during adult life for exercise and to recover from injuries and diseases. Defects in the genetic programming underlying these processes give rise to myopathies. The molecular mechanisms underlying muscle growth are not fully elucidated, but it is known to be triggered by the initial contact between neuron and muscle fiber. This interaction promotes secretion of agrin by the motor neuron to initiate development of neuromuscular junctions. However, recent studies suggest that agrin may play a more prominent role in skeletal muscle growth and development. In this thesis, I characterized this new role for agrin by studying model myoblasts (cultured C2C12 cells) using fluorescence microscopy, immunoblot analysis and qPCR analysis. First, I examined effects of agrin on muscle cell proliferation and differentiation by treating C2C12 cells with recombinant neural agrin for the duration of their differentiation into myotubes. Agrin-treated cells show greater levels of MEF2c and myogenin, markers indicative of differentiation, which correlates with an observed increase in myotube number and area. I then determined that agrin did not use the calcineurin pathway to achieve these phenotypes. Rather, agrin was able to restore growth of calcineurin-inhibited cells, suggesting it targets a different second messenger system. As the Akt/mTOR pathway is critical for muscle growth, I tested whether agrin utilizes this signaling pathway through the use of an mTOR inhibitor, rapamycin. Although the Akt/mTOR pathway was found to be upregulated with agrin treatment, this did not fully account for the effects of agrin on growth and differentiation, suggesting that perhaps another second messenger pathway may be responsible. Although there is much more to explore, this data uncovers a new role for agrin in the development of the early stages of skeletal muscle growth. These new findings allow a better understanding of muscle physiology, and serve as a novel potential target in the treatment of sarcopenia and cachexia.