Domain specific over-expression of a peptide encoded by an I-band domain of the human TTN gene; the role of titin exons 248 – 250 in C2C12 myogenesis

Abstract

In 1996, a form of Rippling Muscle Disease (RMD) was characterized as a heterogenic autoimmune neuromuscular disease. The mechanics of the main symptom, rolling contractions along the length of a muscle induced by stretching or tapping, are not yet understood, however, studies have suggested that the gigantic skeletal muscle protein titin may play a potential role. We hypothesize that an overexpression of a specific immunogenic titin domain would have an effect on the natural processes of cell growth, proliferation, and differentiation of C2C12 Mus musculus myoblast cells. A fusion plasmid was constructed to produce a fusion protein incorporating the immunogenic titin domain attached to a green fluorescent protein. C2C12 cultures were then transfected with the fusion plasmid and induced into differentiation over a two week period. Morphology and fluorescent patterns were analyzed using light and fluorescent microscopy techniques. Protein samples taken at various time points throughout the differentiation process were analyzed using SDS-PAGE. Techniques of the analysis of in vivo production of the immunogenic titin domain were developed. This preliminary data suggests that over-expression of an immunogenic titin domain has an effect on normal growth and differentiation of C2C12 cells. We have also shown that this method of study can be used to identify roles of not only this but other domains of the titin protein.