Figure 4. Muscle inactivity down-regulates the TOR pathway and induces eIF4EBP3L mRNA.

Wild-type (A–C, E) or chrnd^sb13/+ incross zebrafish embryos were incubated with (grey bars) or without (black bars) MS222 for 17–24 h. At 48 hpf, embryos were analyzed by Western analysis (A), immunostaining (B), or qPCR relative to actin (20 embryos/sample; C). (A and B) Muscle inactivity reduces TORC1 activity. Phosphorylation levels of downstream TORC1 targets were reduced both in whole embryo (A) and in muscle tissue (B). Scale = 200 µm. Note that both eIF4EBP antisera likely detect eIF4EBP1, 2, and 3L as the epitope sequence is conserved between human and zebrafish (see Figure S4C). (C and E) Electrical inactivity specifically increased eif4ebp3l and eif4ebp1 mRNA in whole embryos (C). Several zebrafish E3 ligase atrogenes showed similar increases in mRNA level (E). (D) Specific loss of muscle activity in acetylcholine receptor δ mutants (identified by their immotility) induces eif4ebp3l, but not eif4ebp1 mRNA, compared to siblings. Actin served as a control (A, C–E) for normalization. Error bars represent SEM and samples were compared by t test. All experiments were repeated at least twice.