Tricaine blocks muscle contraction at high concentrations.

<p><b>A.</b> Comparison of tail displacement (<i>d</i>) as a function of successively increasing tricaine concentration in <i>fixe</i> mutants and their siblings. In both cases, full movement is regained upon tricaine wash-out to fish water. <b>B.</b> Tail displacement in response to L-type Ca_v channel blocker Nifedipine alone, tricaine alone, or both, and after wash-out of both drugs (right hand bar). Error bars are SEM. Numbers of embryos are shown on bars.</p

Tricaine fails to block evoked muscle contractility.

<p><b>A.</b> The electrical stimulation set-up (ESS). Fish are placed within the central well aligned, when spontaneous motility permits, with their anteroposterior axis perpendicular to the electrodes. Schematic of the electric stimulus regime is shown below. <b>B-D</b>. Tail displacement (<i>d</i>) in embryos from wild type (B) or a <i>fixe</i> heterozygote incross sorted into motile siblings (C; <i>chrnd⁺</i>^/<i>?</i> 97/132 = 73.5%) and immotile mutants (D; <i>chrnd^sb13</i>^/<i>sb13</i> 35/132 = 26.5%). Movement was quantified from videos as measured displacement (<i>d</i>) of tail as shown (B-D, right panels) in superimposed stills of single fish before stimulation (black image) and at maximum displacement (false-coloured red image). Unanaesthetized motile fish move extensively, generally out of the field of view (indicated by break in <i>Y</i>-axis). Fish were measured before tricaine exposure, after exposure to 0.61 mM tricaine for 30 min and after ≤30 minutes of tricaine washout into fish water. Note altered <i>Y</i>-scale in D compared to B,C. <b>E.</b> Comparison of mutants with tricaine-treated individuals reveals a striking similarity in displacement. Error bars are SEM. Numbers of embryos are shown on bars. Scale bars  = 0.1 mm.</p

Active eIF4EBP3L regulates myofibrilogenesis following inactivity.

<p>Embryos were injected with the indicated MO, some exposed to MS222 for the last 17(A) or with plasmid DNA encoding a constitutively active eIF4EBP3L and GFP (5A3L-GFP) or empty vector (GFP) (B) were grown to 48–54 hpf and immunostained for slow MyHC. (A) BP3L MO1 prevents, and BP3L MO2 reduces, the decrease in slow MyHC caused by inactivity. Immunostaining (left panels) was quantified by confocal microscopy (right panel; <i>n</i> = 15 embryos/sample). (B) Overexpression of 5A3L reduces myofibril bundle width. Following heat shock, the size of slow myofibril bundles in GFP+ fibers was determined relative to their GFP− neighbors (<i>n</i> = 9 embryos/sample). Bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice. Scale bar = 23 µm.</p

Muscle activity enhances zebrafish myofiber width and myosin level.

<p>Muscle activity was blocked either by adding MS222 to the fish water for 24<i>chrnd</i>^sb13/sb13 mutants lacking the acetylcholine receptor delta subunit, which were identified by their immotility. (A) Confocal stacks of slow MyHC immunostaining in 48 hpf embryos. Note the reduced myofibril content and poor bundling in inactive fish. White bars in the right-hand panels indicate minimal myofibrillar bundle width on each fiber. Fivefold more laser light was used to generate the lower right image. (B) Width of myofibrillar material in well-bundled regions of >5 myofibers in somite 17 were measured from >6 embryos in each condition. (C) Slow MyHC level relative to control in <i>n</i> = 15 embryos. (D and F) Western analysis of 48 hpf mutant or MS222-treated embryos, compared to respective controls. (E) Confocal stacks of embryos stained for general MyHC (A4.1025) in lateral (left image) and transverse (right image, somite indicated by white line) view. Graph shows relative MyHC level. <i>n</i> = 10 embryos. Bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice. Scale bar = 90 µm in (A, left), (E), and 23 µm in (A, right).</p

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

<p>Wild-type (A–C, E) or <i>chrnd^sb13/+</i> 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 <i>actin</i> (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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679.s004" target="_blank">Figure S4C</a>). (C and E) Electrical inactivity specifically increased <i>eif4ebp3l</i> and <i>eif4ebp1</i> 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 <i>eif4ebp3l</i>, but not <i>eif4ebp1</i> mRNA, compared to siblings. Actin served as a control (A, C–E) for normalization. Error bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice.</p

Muscle activity regulates translation of <i>mef2ca</i> mRNA.

<p>Zebrafish embryos were exposed to MS222, Rapamycin <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679-Drapeau1" target="_blank">[34]</a>, or vehicle control. (A and B) Muscle activity does not regulate Mef2 protein via the proteasomal or autophagic pathways. (A) Western blot of whole embryos (48 hpf; 50/sample) incubated with 100 nM MG132 or DMSO vehicle for 1 h prior to and then during 17 h MS222 treatment. (B) Quantification of Mef2c and full-length 50 kd Sqstm1 bands relative to Actin. (C) qPCR of the indicated mRNAs relative to <i>actin</i> following MS222 or vehicle (48 hpf 20 embryos/sample). (D) Schematic of separation of cytoplasmic extract on a sucrose gradient into light sub-polysome (SP) and heavy polysome (P) fractions to reveal fraction of mRNA in each. (E) Polysomal profiles of nucleic acid from the gradient of active control and inactive MS222 zebrafish. Peaks indicate 40 s, 60 s subunits, and 80 s monosomes. The amount of nucleic acid in the polysome (P) and subpolysome (SP) fractions were calculated from the areas between the dashed lines. (F) Differential regulation of muscle mRNAs by activity. Proportion of each specific mRNA in the P fraction, SP fraction, and unfractionated (total) was measured by qPCR. Activity-dependent change in translation rate at 48 hpf was determined as the ratio of polysomal/total in MS222 to polysome/total in control (70 embryos/sample). The level of each gene in the fraction was normalized to its level in the total to rule out change in transcription. To present the average from several experiments, the translation change was normalized to the relative change in <i>mef2ca</i>. Bars represent SEM (<i>n</i> = 4) and samples were compared by <i>t</i> test. All experiments were repeated at least twice.</p

Model of how muscle activity stimulates muscle growth through differential translational regulation.

<p>Previous studies indicated a role of TORC1 (a known cell size regulator) in activity-dependent muscle growth (1). TORC1 regulates growth by inhibiting the translation initiation inhibitor eIF4EBP (2) and by activation of S6K (3) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679-Ishikawa1" target="_blank">[108]</a>, both leading to increased translation. In the absence of muscle activity, muscle fibers undergo atrophy, a reduction in size and strength. Both the FoxO/Atrogin-1 axis (4) and the calcineurin pathway (5) contribute to atrophy. We found that during muscle development lack of muscle activity increases the levels of eIF4EBP3L in muscle (6) and reduces the activity of TORC1 pathway (1), preventing eIF4EBP3L phosphorylation (2) and thereby activating it. High levels of active eIF4EBP3L prevent initiation of protein synthesis from specific mRNAs (7). Among mRNAs regulated by TORC1/eIF4EBP3L is that encoding the transcription factor Mef2ca, which is also regulated by calcineurin and is required for normal myofibrilogenesis and muscle growth (8). We hypothesize that other mRNAs are also differentially regulated by the activity/TORC1/eIF4EBP3L axis (9) and contribute to muscle homeostasis along with the FoxO and calcineurin pathways.</p

Overexpression of eIF4EBP3L reduces and knockdown rescues Mef2c.

<p>Zebrafish embryos injected with RNA encoding eIF4EBP3L (A–C, F) or control MO or BP3L MO1 (D, E) were grown to 48–54 hpf and some exposed to MS222 for the last 17 h (A, B, E) or to 5 µM rapamycin for 6 h (Rap; A, C, F) or remained as untreated controls. (A–C) Mef2c immunostaining intensity quantified by confocal microscopy in 54 hpf embryos shows that overexpression of eIF4EBP3L reduced Mef2c protein in inactive (B) or rapamycin-treated (C) muscle. <i>n</i> = 60 (B) and <i>n</i> = 20 (C) embryos/sample. Scale bar = 90 µm. (D) Level of indicated mRNAs assayed by qPCR shows that BP3L morphants have reduced <i>eif4ebp3l</i> mRNA. (E) Western analysis shows that BP3L MO1 prevented decrease in Mef2c caused by inactivity compared to actin loading control. Bars represent SEM. Samples were compared by <i>t</i> test. All experiments were repeated at least twice. (F) Polysomal fractionation followed by triplicate qPCR for <i>mef2ca</i> and <i>dmd</i> mRNAs on polysomal and subpolysomal fractions.</p

Notch signalling is crucial for skeletal muscle integrity and stress fibres formation.

<p>Immunostaining of 5 dpf pectoral fins (A–G”) with DAPI and phalloidin to label nuclei (blue) and filamentous actin (green), respectively. Stress fibres in the endoskeletal disc cells and intact skeletal muscle fibres are formed in the pectoral fins of siblings (n = 20) (A–A”) and in DMSO-treated embryos (n = 17) (F-F”). In the pectoral fins were Notch signalling was disrupted like in <i>mib</i>^ta52b (n = 22) (B–B”) and <i>mib</i>^m178 (n = 10) (C–C”) mutants, <i>jagged2</i> (n = 11) (D–D”) and <i>Su(H)</i>1+2 (n = 10) (E–E”) morphants and in DAPT-treated embryos (n = 16) (G–G”) the endoskeletal disc cells present high levels of actin at the periphery and the skeletal muscle fibres are wavy with gaps between them.</p

Mechanical weak muscles fibres are produced when Notch signalling is perturbed.

<p>Transmission electronic microscopy was performed in 5 dpf pectoral fins of siblings (A) and <i>mib</i>^ta52b mutants (B) to analyse the ultrastructure of skeletal muscle fibres. The myofibrils with clear aligned sarcomeres are formed in the sibling embryo (A), while disintegrating myofibrils with poorly aligned sarcomeres are found in <i>mib</i>^ta52b mutants (B). Immunostaining performed in pectoral fins at 5 dpf using Desmin, Vinculin, phalloidin and DAPI to label the Z-discs of the sarcomeres, the zone where the skeletal muscle fibres insert distally, filamentous actin and the cell nuclei, respectively (C–P) demonstrate that Desmin (n = 10) (D) and Vinculin (n = 12) (F) are downregulated in <i>mib</i>^ta52b mutants when compared with their siblings (n = 10) (C, E). The same is observed in DAPT-treated embryos at 48 hpf and fixed at 5 dpf (n = 9) (H), when compared with the DMSO-treated embryos (n = 10) (G). In embryos treated with blebbistatin, Desmin (n = 9) and Vinculin (n = 12) are also downregulated (J, L) when compared with the DMSO-treated embryos (n = 12) (I, K). In blebbistatin-treated embryos, the endoskeletal disc cells present high levels of actin at the periphery and the skeletal muscle fibres are wavy with gaps between them (n = 15) (N). A down-regulation of Desmin and Vinculin is also observed in MS222-treated embryos (n = 14) (P) when compared with the control embryos (n = 13) (O).</p