Expression of MuSK in in vitro-innervated human muscle

Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.