Chracterisation of Mighty during Skeletal Muscle Regeneration

Abstract

Satellite cells are a distinct lineage of myogenic precursors that are responsible for the growth of muscle during post-natal life and for its repair after damage. During muscle growth and regeneration satellite cells are activated in response to growth signals from the environment, which induces the expression of one or both of the two MRFs, Myf-5 or MyoD. Activated satellite cells migrate to the site of injury and proliferate before these transcription factors go on to activate transcription of myogenic genes. The myoblasts can then adopt one of two fates. Some myoblasts initiate terminal differentiation and are able to either fuse into existing myofibres to repair them, or fuse with other myoblasts to form new fibres. Other myoblasts do not differentiate but instead return to quiescence and adopt a satellite cell position on repaired or newly formed fibres.

Mighty, a downstream target of myostatin that was discovered by the Functional Muscle Genomics Laboratory has recently been shown to induce cell hypertrophy in cell culture through enhanced differentiation and fusion of myoblasts. Myostatin-null mice have hypertrophic muscles and an improved muscle regeneration phenotype. These mice have also been shown to have higher basal levels of Mighty in skeletal muscle than wild-type mice. In this thesis the expression profile of Mighty during skeletal muscle regeneration was characterised in relation to MyoD.

During regeneration Mighty gene expression was induced at day five post-injury in both wild-type and myostatin-null mice. In the myostatin-null mice Mighty gene expression remained elevated at day seven post injury in contrast to the levels in the wild-type, which had decreased at this time point. By day-14 and day-28 post-injury Mighty levels were decreased. The up-regulation of Mighty occurs at the time of peak myotube formation in regenerating skeletal muscle, consistent with a role for Mighty in enhancing differentiation and fusion of myoblasts. The extended up-regulation of Mighty in the myostatin-null muscle may be responsible for the enhanced regeneration phenotype of these mice.

Analysis of the myotube and reserve cell populations, which are an in vitro model of satellite cells, from both C2C12 cells and Mighty over-expressing clones (Clone 7 and Clone 11) showed that Mighty expression down-regulates two satellite cell markers, CD34 and Sca-1. Both these molecules have been recently shown to be involved in myoblast fusion and reserve cell specification, although their exact role in these processes is not yet known. Expression of Sca-1 is associated with a slowly proliferating non-dividing state while CD34 is associated with the population of reserve cells that do not fuse when notch signalling is inhibited. The results of this thesis indicate that Mighty over-expression may cause the enhanced fusion phenotype by regulating these two molecules.

In conclusion the data in this thesis supports a role for Mighty in the myotube formation phase of regeneration and may be able to enhance regeneration by recruiting more myoblasts to terminal differentiation by altering CD34 and Sca-1 expression.