Nkx2-5 is an important transcriptional regulator of cardiogenesis, required for the correct patterning of the heart and the maturation of the cardiac chambers. A differential screen for genes downregulated in homozygous Nkx2-5 knockout mouse hearts revealed disruption of Chisel (Csl) gene expression. Csl encodes a 9kDa protein that is strongly expressed in mature striated muscle. This thesis investigates Csl function by a variety of experimental approaches.

The heat shock protein Hsp70 and an activated form of the calcium-dependent phosphatase calcineurin interacted with Csl protein in an in vitro binding assay. Hsp70 has multiple intra- and extra-cellular functions, but in muscle it is strongly upregulated following stress. Similarly, calcineurin has been proposed to be a key regulator of stress and growth factor-induced muscle hypertrophy. Immunohistochemical staining using a Csl-specific antibody revealed that Csl protein localised to the muscle costamere, adjacent to the Z-line. This unusual pattern of distribution is shared by several muscle- enriched proteins, ablation of which produces no detrimental effects on striated muscle formation, but results in a reduced capacity to cope with muscle stress. Thus we propose that Csl may also function in the muscle stress response. In concert with insulin-like growth factor-I treatment, Csl overexpression induced promiscuous fusion and cytoskeletal disorganisation in differentiating C2C12 myotubes. Many of the proteins that localise to the muscle costamere can also influence cell spreading in motile cells. Likewise, enforced expression of Csl in C2C 12 myoblasts was found to enhance cell spreading and lamellipodia extension. Moreover, Csl induced a fundamental shift from filopodia to lamellipodia formation in spreading myoblasts that was independent of changes in overall actin turnover. Inhibitor studies indicated that Csl-induced spreading is dependent on a Racl-p38 kinase pathway previously implicated in lamellipodia extension and stress responses. Csl is a phosphoprotein that is differentially phosphorylated between cardiac and various skeletal muscles. Although we have speculated that Csl functions in response to muscle stress, stress did not alter Csl phosphorylation. In conclusion we propose that Csl is a component of a specific cytoskeletal sub-domain, the Z-line adjacent costameric cytoskeleton, and that this sub-domain plays an integral role in muscle regeneration and repair.