|dc.description.abstract||Myoblast fusion is a dynamic event that can be recreated in vitro using tissue culture. Portions of cDNA encoding inhibitory and non-inhibitory domains of ovine calpastatin were expressed in murine C2C12 myoblasts as fusion proteins with enhanced green fluorescent protein (EGFP). The ability of these myoblasts to fuse into myotubes, the dynamics of particular indicator proteins, and the subcellular localisation of fluorescence were assessed.
Formation of skeletal muscle is a multistage process characterised by the biochemical and morphological maturation of myoblasts (mononucleate) into myotubes (multinucleate). A key event in the transition from myoblast to myotube is fusion. Fusion is a dynamic event which can be induced and monitored by culturing muscle cells in vitro. An expanding body of evidence indicates that proteolytic activity from the ubiquitous calpains (µ- and m-) is obligatory for fusion and myotube development.
Calpastatin is the specific endogenous inhibitor of calpain. Domains 1, 2, 3 and 4 are homologous and possess inhibitory potential. The N-terminal domain L may playa role in regulating calpastatin action and / or directing the localisation of calpastatin to the plasma membrane. Proteolytic activity is prevalent around the plasma membrane during fusion. It was decided to investigate domain L's potential role in targeting calpastatin to this region.
The polymerase chain reaction (PCR) was used to amplify regions of ovine calpastatin cDNA corresponding to domains L, 1 and L1. The PCR reaction enabled the introduction of restriction enzyme sites to permit in-frame ligation of the amplimers into a prokaryotic expression vector. Using E. coli, domains L and 1 of ovine calpastatin were expressed separately and together, in combination with glutathione S-transferase (GST). The GST tag enabled purification of the expressed product which was then characterised in terms of heat stability, molecular weight and activity. The proteins behaved normally in respect to their ability to inhibit calpain, and were used as antigens for the successful production of polyclonal anti-calpastatin antibodies.
C2C12 myoblasts were cultured under proliferation conditions before being induced to fuse. The rate and extent of fusion in control cells was assessed by microscopic examination and by fluorescent automated cell sorting (FACS). FACS analysis was unable to accurately distinguish myotubes and significantly underestimated the percentage fusion at all times. Three proteins (myosin, desmin and troponin T) were reported to be indicators of biochemical differentiation while desmin and troponin T are also potential substrates for calpain. Levels of these proteins and patterns of migration were determined using Western analysis. While the levels of all three proteins reflected biochemical differentiation, only troponin T Westerns exhibited truncation products. It could not be concluded if this truncation was associated with calpain activity. Levels and fragmentation patterns of µ-calpain, m-calpain and calpastatin were also assessed. Both proteases were detected in C2C12 extract in an active form. Despite one of the polyclonal anti-calpastatin antibodies demonstrating immunoreactivity against native murine calpastatin, no endogenous calpastatin was detected in C2C12 extract.
The polymerase chain reaction (PCR) approach used to produce prokaryotic expression vectors was also used to produce eukaryotic expression vectors. Amplimers of domains L, 1 and Ll were ligated into a mammalian expression vector containing EGFP cDNA. Conditions for transfection of C2C12 myoblasts were optimised using a control EGFP expressing plasmid. Cells were transfected with one of four expression plasmids (three experimental plasmids and one control), encouraged to proliferate and induced to fuse. There were no differences in terms of fusion rate or extent attributable to the expression of calpastatin. FACS analysis did not
reveal any qualitative differences in terms of fluorescence between treatments.
Western blot analysis of myosin, desmin, troponin T, µ-calpain and m-calpain did not detect any qualitative or quantitative differences. Western analysis of the expressed calpastatin products revealed complex banding patterns which may reflect structural instability and / or proteolytic susceptibility. A schematic model is proposed which seeks to depict the truncation points.
Finally, fluorescent microscopy and confocal microscopy were used to localise the fluorescent component of the recombinant calpastatin - EGFP hybrids. Examination of transfected cells indicated that the active component of the expressed calpastatin was localised, together with EGPF, within the nucleus. In contrast, domain L did not influence the subcellular localisation of EGFP, although this may be explained by the probable separation of domain L from EGFP suggested in the schematic model.||en