Caveolin-3 is a muscle specific scaffolding protein with both structural and signaling roles. Lack of caveolin-3 expression has been implicated in limb-girdle muscular dystrophy, along with distal myopathy and rippling muscle disease. These diseases are characterized by progressive muscle weakness and muscle wasting. Nicotinic acetylcholine receptor (nAChR) clustering and localization are important for efficient nerve to muscle contractile signal transmission. It is hypothesized that muscle weakness could originate through disrupted nAChR clustering, disrupting the efficiency of signaling from the motorneuron to the muscle. While the molecular mechanisms involved in nAChR clustering remain to be fully defined, we hypothesize caveolin-3 is important for nAChR clustering and overall neuromuscular junction function.Caveolin-3 and the nAChR co-localize and associate evidenced by immunofluorescence and immunoprecipitation. These results were replicated in differentiated wildtype myotubes treated with the nAChR clustering agent, neural agrin. In differentiated caveolin-3 null myotubes, agrin treatment yields a 60% reduction in nAChR clusters as compared to agrin treated wildtype myotubes. Agrin induces nAChR clustering, through activation of muscle specific kinase (MuSK) and downstream through Rac-1 activation. In differentiated wildtype myotubes, Rac-1 activation peaks at 1 hour of agrin treatment, while in differentiated caveolin-3 null myotubes there is dramatically reduced Rac-1 activation upon agrin treatment. Immunoprecipitation of MuSK shows that caveolin-3 and MuSK association peaks at 1 hour of agrin treatment in wildtype cells. This corresponds to the peak of MuSK phosphorylation which also occurs at 1 hour. Agrin induced MuSK phosphorylation was decreased more significantly than the overall decrease in MuSK expression in the caveolin-3 null cells as compared to the wildtype results. These results indicate a role for caveolin-3 in efficient nAChR clustering.Electromyography studies in anesthetized mice indicated lengthened latencies of the muscle action potential in the caveolin-3 null mice as compared to wildtype mice. There were also decreased overall electromyography (EMG) amplitude and EMG area under the curve in caveolin-3 null mice. Comparison of contractile strength in wildtype and caveolin-3 null animals indicated tetanic contractions to be less stable in the caveolin-3 null animals, though there was late potentiation in actual contractile strength.Lack of caveolin-3 affects the neuromuscular junction formation and transmission without affecting overall contractile strength. This research opens a novel view, that correct neuromuscular junction formation and neuromuscular transmission is important in the development of muscular dystrophies
