Aim: 

Clusters of embryonic muscle nAChRs form in the absence of neurons during the development of skeletal muscle cells. Autocrine activation of nAChR channels contributes to the spontaneous oscillations of intracellular concentration of Ca²⁺([Ca²⁺]_i) and to twitches characteristic of developing myotubes before innervation. The aim of the present study was to test if the aggregation of the nAChRs could modulate the Ca²⁺ spiking activity during myogenesis.

Methods: 

Mouse i28 myoblasts derived from satellite cells were differentiated in vitro up to 12 days. The nAChR pattern distribution was analysed by confocal microscopy. The [Ca²⁺]_i was measured by videoimaging and the single channel properties of nAChRs by patch clamp.

Results: 

Clusters of nAChRs were observed starting from day 2 of differentiation. Their number was the highest after 11 days, when they were detected in 39.85 2.59% of the cells (1 per cell; mean length 11.76 0.50 mm). The nAChR labelling showed that 75.75 3.87% of the cells with clusters exhibited spiking and twitching. A 24 h incubation with STI 571 10 mM, a specific inhibitor of Abl (nonreceptor tyrosine kinases which control the nAChR clustering) significantly decreased the number of cells with clusters (to 13.39 2.40%) and the number of spiking and twitching cells (from 23.95 1.81% to 7.40 0.91%). Patch clamp recordings in the cell attached configuration showed that this pharmacological treatment did not affect amplitude and kinetics of the nAChRs.

Conclusion: 

Our results indicate that the formation of nAChR clusters could favour spontaneous Ca²⁺ spiking and twitching during myogenesis. These data suggest a possible new role for the so-called "prepatterned AChR clusters" before the arrival of the nerve.