Regulation of Acetylcholinesterase (AChE) mRNA by Ryanodine-Sensitive and L-Type Calcium Channels during Myogenesis in Vitro and Muscle Development in Vivo

Abstract

The increase in AChE mRNA associated with muscle differentiation is due to stabilization of a labile mRNA rather than enhanced transcription (Fuentes and Taylor, Neuron, 10, 679–687, 1993). In a search for the signaling pathways responsible for message stabilization, we examined the roles of ryanodine-sensitive and L-type Ca²⁺ channels in regulation of AChE mRNA during myogenesis. Treatment of C2-C12 cells in cultures during myogenesis with nM ryanodine, μM ryanodine receptor antagonists, FLA 365, and μM L-type, but not N-type, Ca²⁺ channel antagonists blocked the differentiation-induced increase in AChE mRNA and protein in a dose dependent manner. Ryanodine and the L-type Ca²⁺ channel blockers did not influence the enhanced expression of the nicotinic acetylcholine receptors (nAChR) associated with fusion. The ryanodine block was fully reversible within 24 hours upon removal of the drug indicating the functional integrity of the cells. Measurements of transcription rates using run-on transcription showed that ryanodine and L-type Ca²⁺ channel antagonist, nifedipine, did not change the rate of AChE gene transcription indicating that the block in the increased mRNA associated with differentiation was due to reduced stabilization of the labile mRNA. These findings indicated that ryanodine-sensitive Ca²⁺ channels in sarcoplasmic reticulum and L-type Ca²⁺ channels in T-tubules of skeletal muscle link to play important roles in regulation of AChE mRNA during myogenesis (Luo et al., J. Biol. Chem., 1994, in press). To confirm the importance of this signaling pathway in AChE regulation in intact skeletal muscle, we examined AChE mRNA levels in skeletal and cardiac muscles from muscular dysgenic mice lacking the skeletal, but not the cardiac, L-type Ca²⁺ channel receptors. RNA protection experiments indicated 50–80% reductions in AChE mRNA levels in leg muscles from new born and day 18 embryonic mutant mice as compared to control mice. Similar reductions in AChE activity were also observed. However, no reduction in mRNA levels of nAChR y-subunit was observed in leg muscles and mRNA levels and AChE activity were not altered in cardiac tissues from mutant mice. These findings provide evidence to indicate that L-type Ca²⁺ channels also play an important role in regulation of AChE expression in intact skeletal muscle. The differential regulation of mRNA levels of AChE and nAChR suggests distinct mechanisms of regulation controlled by L-type Ca²⁺ channels in intact skeletal muscles.