Differentiation of C2C12 cells results in enhanced expression of δ Ca2+/calmodulin‐dependent protein kinase II isoforms

Abstract

Previous work from our lab provided evidence for increased expression of δ-isoforms of calcium/calmodulin-dependent protein kinase II (CaMKII) in skeletal muscle undergoing regeneration after spontaneous or digitonin-induced necrosis. C2C12 myoblasts were used to examine how CaMKII expression changes during skeletal muscle differentiation. These cells were cultured in 10% fetal bovine serum-containing media and then allowed to differentiate into myotubes for up to 4 days. Western blots of extracts from these cells were probed with a pan antibody against CaMKII, which detected two CaMKII isoforms (50, 51 kDa) in undifferentiated myoblasts. Within 24 hours of differentiation there was a 2–3 fold increase in CaMKII expression that remained elevated during the 4-day period. Furthermore, differentiating cells were found to express 2–3 additional CaMKII bands in the 48–56 kDa range. Probing these samples with a δCaMKII-selective antibody suggested that these expressed proteins were most likely δ-isoforms. RT-PCR of RNA from these cells, using specific primers against the variable regions δCaMKII, produced multiple amplicons that were consistent with the upregulation of δCaMKII isoforms. Confocal imaging of cells labeled with CaMKII antibodies, demonstrated that immunoreactivity to these antibodies was primarily associated with differentiated myofibers rather than myoblasts. These results suggest that differentiating skeletal muscle express multiple isoforms of δCaMKII that may play a critical role in the differentiation/regeneration process.