The first step was to examine the dynamics of MuRF expression in the zebrafish larvae in relation to contractile function, using dexamethasone treatment known to up-regulate MuRF expression in skeletal muscle [10]. We treated larvae with 300 µM dexamethasone from 3 to 5 dpf, and found a significant up-regulation of MuRF-1 and a lower active force generation as shown in Fig. 1. To explore if pharmacological immobilization affected MuRF-1 expression and active force we immobilized zebrafish larvae from 3 to 6 dpf using an actomyosin inhibitor (BTS) to inhibit contractile function and active swimming completely. Although the larvae stopped swimming and become unresponsive to gentle touch, normal heart beating and blood circulation were observed by microscopy. After removal of BTS prior to mechanical analysis, larvae swimming recovered in all the groups within 40 min, and therefore a 60 min BTS-wash out time period was used to ensure that no residual BTS could influence subsequent mechanical analysis. Maximal active force at optimal length was determined at different developmental and BTS immobilisation time. Immobilization had significant effect on active force (Table 2).
Table 2
Summary of p values from Two-way ANOVA
|
Maximal active force
|
MuRF-1
|
MuRF-2
|
MuRF-3
|
MAFbx
|
Days
|
0.458
|
0.143
|
0.197
|
0.229
|
< 0.001
|
Immobilization
|
< 0.001
|
0.016
|
0.001
|
0.034
|
0.022
|
As shown in Fig. 2, active force increased gradually with development from 4 to 6 dpf in DMSO-treated controls. In the immobilized group, active force was slightly lower but not significantly different compared to the controls after 1 day of immobilization (at 4 dpf). However, the difference became bigger after 2 days of immobilization (at 5 dpf). Immobilized larvae were significantly weaker compared to the controls at 5–6 dpf as illustrated in Fig. 2. To see if the effect of immobilization on active force is reversible, larvae treated with BTS for 1 day were transferred to BTS-free medium for recovery. After 2-day recovery (from day 4 to 6 dpf), active force was similar to that in the controls at 6 dpf (hatched bar compared to open bar at 6 dpf, Fig. 2). In contrast, larvae immobilized for 2-days and recovered only for 1-day did not recover (grey bar compared to open bar at 6 dpf, Fig. 2).
The expression of MuRF-1 to 3, and MAFbx/atrogin-1 were altered due to immobilization during 4–6 dpf as summarized in Table 2. As shown in Fig. 3A, MuRF-1 had the highest expression among these four genes, whereas MuRF-3 expression was the lowest in general. In the controls, the expression of the MuRF-1 and MAFbx increased gradually during muscle development from 4–6 dpf. After 1-day immobilization, no difference was noticeable in any of the examined ligases expression. There is a tendency that immobilization reduced ubiquitin ligase expression as illustrated in Fig. 2A. After 1-day immobilization (at 5 dpf), compared to the controls, MuRF-1 and − 2 expression became significantly lower. After 3-day immobilization, MuRF-2, -3 and MAFbx expression was significantly lower in treated groups compared to the controls. To examine if immobilization would induce more rapid (within hours) changes in MuRFs and MAFbx, control larvae at 5 dpf were immobilized with BTS for only 1, 2 and 4 hours. As seen in Fig. 3B, no changes were observed in these genes in this shorter time perspective. In a separate series we dechorionated the larvae at 18 h post fertilization, and thereafter immobilized them until 4–6 dpf. In this circumstance where larvae never moved until being analysed, the changes of ligase expression were similar compared to the ones immobilized from 3 dpf. In the controls, MAFbx expression increased significantly from 4–6 dpf (Table 2), which correlated with muscle development.
To explore that if the immobilization affected the contractile filament structure, we performed x-ray scattering experiments. Strong scattering patterns were recorded from 6 dpf larvae with clear 1.1 and 1.0 spacings of about 25 and 43 nm respectively as shown in Fig. 4. Panel A and B show the equatorial x-ray pattern from 6 dpf controls and 6 dpf larvae immobilized from 3 dpf. A clear outward movement of 1.0 and 1.1 reflections was observed after 3-day immobilization, demonstrating the shrinkage of the filament lattice. The average spacing of the 1.0 reflection (d1.0) from multiple analysis was significantly smaller in the BTS treated group (Fig. 4C).