Heat-shock protein 90alpha1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos

Abstract

Heat-shock protein 90alpha (Hsp90alpha) is a member of the molecular chaperone family involved in protein folding and assembly. The role of Hsp90alpha in the developmental process, however, remains unclear. Here we report that zebrafish contains two Hsp90alpha genes, Hsp90alpha1, and Hsp90alpha2. Hsp90alpha1 is specifically expressed in developing somites and skeletal muscles of zebrafish embryos. We have demonstrated that Hsp90alpha1 is essential for myofibril organization in skeletal muscles of zebrafish embryos. Knockdown of Hsp90alpha1 resulted in paralyzed zebrafish embryos with poorly organized myofibrils in skeletal muscles. In contrast, knockdown of Hsp90alpha2 had no effect on muscle contraction and myofibril organization. The filament defects could be rescued in a cell autonomous manner by an ectopic expression of Hsp90alpha1. Biochemical analyses revealed that knockdown of Hsp90alpha1 resulted in significant myosin degradation and up-regulation of unc-45b gene expression. These results indicate that Hsp90alpha1 plays an important role in muscle development, likely through facilitating myosin folding and assembly into organized myofibril filaments.

Fig. 1.

Gene structure and sequence comparison of zebrafish Hsp90α1 and Hsp90α2. (A) Zebrafish Hsp90α1 and Hsp90α2 share similar gene structures with 11 exons and 10 introns. Open boxes represent the 5′- and 3′-UTR regions. (B) Sequence comparison showing the region with more diverged sequences in Hsp90α1 and Hsp90α2 proteins. Sequence alignment indicates that zebrafish Hsp90α2 is more closely related to mouse and human Hsp90α. (C) MO antisense oligos and their target sequence in Hsp90α1 and Hsp90α2. The ATG start codon is identified in red. The intron sequences are shown in lowercase letters.

Fig. 2.

Temporal and spatial expression of Hsp90α1 and Hsp90α2 in zebrafish embryos. (A–D) Muscle-specific expression of Hsp90α1 in somites and skeletal muscles at 13 (A and B) and 24 hpf (C and D). (A and B) Dorsal view of anterior (A) and posterior (B) regions of a 13-hpf embryo. (C and D) Side (C) and dorsal (D) view of a 24-hpf embryo. The enzymatic reaction was carried out for 3 h during the whole-mount in situ hybridization. (E and F) Longer in situ staining (12 h) shows the cardiac expression of Hsp90α1 (arrow in E) in comparison with the cardiac muscle specific expression of myosin light chain (F). A weak staining of Hsp90α1 was also found in the brain and eye regions (E). (G and H) Cross (G) and horizontal (H) sections showing the muscle-specific expression of Hsp90α1 in zebrafish embryos at 24 hpf. (I–L) Expression of Hsp90α2 in somites, skeletal muscles and other tissues at 13 (I and J) and 24 hpf (K and L). In addition to muscle expression, Hsp90α2 expression was also detected in the head and eye regions (I). The enzymatic reaction was carried out for12 h during the whole-mount in situ hybridization. (Scale bars: A, 250 μm; C, 150 μm; and H, 50 μm.)

Fig. 3.

Knockdown of Hsp90α1 expression resulted in myofibril disorganization in skeletal muscles of zebrafish embryos. (A and B) Anti-MHC antibody (F59) staining shows the organization of thick filaments in trunk slow muscles of control-MO (A) or Hsp90α1-ATG-MO (B) injected embryos at 24 hpf. (C and D) Anti-MHC antibody (F59) staining shows the organization of thick filaments in trunk slow muscles of Hsp90α2 ATG-MO (C) or Hsp90α1-E3I3-MO (D) injected embryos at 24 hpf. (E and F) Anti-actin antibody staining shows the organization of thin filaments in control-MO (E) or Hsp90α1-ATG-MO (F) injected embryos at 24 hpf. (G and H) Anti-α-actinin antibody staining shows the organization of the Z-line in control-MO (G) or Hsp90α1-ATG-MO (H) injected embryos at 24 hpf. (I and J) Anti-myomesin antibody staining shows the organization of the M-line in control-MO (I) or Hsp90α1-ATG-MO (J) injected embryos at 72 hpf. (Scale bars: A and B, 25 μm and I, 15 μm.)

Fig. 4.

Rescue of myofibril organization defect in Hsp90α1 knockdown embryos by ectopic expression of a Hsp90α1 transgene. (A) Western blot analysis shows the expression of myc-tagged Hsp90α1-, Hsp90α2-, or MEEVD-deleted Hsp90α1 (Hsp90α1m) in wild type or Hsp90α1 ATG-MO coinjected embryos. Expression of the myc-tagged proteins was analyzed by Western blot using anti-myc tag antibody (9E10). Each lane contains protein extract from 10 embryos. Actin was used as a loading control. (B) Coimmunoprecipitation shows binding of Hsp90α1, Hsp90α2, or MEEDV domain deleted Hsp90α1 to UNC-45b. (C and D) F59 antibody staining shows the mosaic rescue of myofibers in a Hsp90α1 knockdown embryo co-injected with the smyd1-Hsp90α1 construct (C) or smyd1-GFP vector control (D) at 24 hpf. The rescued myofibers are indicated by arrows. (E and F) Double immunostaining with anti-MHC (E) and anti-myc (F) antibodies shows the rescued myofibers expressing Hsp90α1^myc. (G and H) Double staining with anti-MHC (G) and anti-myc (H) antibodies shows partial rescue in Hsp90α2^myc expressing myofibers. (I and J) Double staining with anti-MHC (I) and anti-myc (J) antibodies shows the partially rescued myofibers expressing the MEEVD domain deleted Hsp90α1. (Scale bars: 30 μm.)

Fig. 5.

Knockdown of Hsp90α1 results in MHC degradation and up-regulation of unc-45b expression. (A) Western blot analysis shows the low levels of MHC proteins in Hsp90α1 knockdown embryos. In contrast, expression levels of actin and myogenin were not affected. (B) RT-PCR analysis of fast-muscle MHC (fMHC), slow-muscle MHC (sMHC), unc-45b, Hsp90α1, and ef-1α expression in control (lanes 1, 3, 5, 7, and 9) or Hsp90α1 ATG-MO injected (lanes 2, 4, 6, 8, and 10) embryos. (C–F) Side views (C and E) or cross-sections (D and F) show unc-45b expression in control (C and D) or Hsp90α1 ATG-MO (E and F) injected embryos at 24 hpf.