We do basic research on the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction and the differentiation of skeletal muscle cells, utilising cell culture systems. Postsynaptic acetylcholine receptor aggregation is a critical early event in neuromuscular junction formation. Agrin is a proteoglycan that is required for normal postsynaptic differentiation in muscle. We previously found that secreted neuronal agrin is consistently concentrated at developing synaptic sites but can be less frequently found on other segments of axons as well as on dendrites or cell bodies. Further, we found that isolated motoneurons initially secrete agrin indiscriminately but progressively accumulate agrin around axons as they mature, indicating a developmentally regulated program for targeting of agrin secretion. We are now attempting to study the intrinsic program for packaging, transport and secretion of agrin in living motoneurons with recombinant agrin-green fluorescent proteins that we have generated. The expression of specific myosin heavy chains (MHCs) is a key indicator of muscle fiber differentiation into the fast or slow phenotype. We have now shown that the synthesis and assembly of slow MHCs is dependent on contractile activity and on the activity of calcineurin. Transfection of myotubes with constitutively active calcineurin markedly upregulates slow MHCs while the calcineurin inhibitor, cyclosporin, blocks their expression. While inhibition of contractile activity by tetrodotoxin inhibits muscle fiber maturation in general, notably the organization of myofibrils, cyclosporin specifically inhibits slow MHC expression, with a concurrent increase in fast/neonatal MHCs. Transfection of primary skeletal muscle cells by non-viral methods is believed to be very inefficient. We have now explored and demonstrated conditions for efficient Transfection of primary myotubes with commercially available transfection reagents. - neuromuscular junction, motoneuron, skeletal muscle, cell culture, rat, tranfection, calcineurin, acetylcholine receptor, agrin, myosin heavy chains
