Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

Abstract

Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.

Fig. 1. Expression of smyhc1 in zebrafish slow muscles and target sequence of smyhc1-MO

A and B: In situ hybridization shows the slow muscle specific expression of smyhc1 in wild type zebrafish embryos at 24 hpf. A, side view; B, view of cross section. C: DNA sequence shows the smyhc1-MO targeted site at the ATG start codon and the intron 1 splicing acceptor. The 5′-UTR, intron 1 and partial coding sequence at the 5′ end are shown.

Fig. 2. Defective splicing of smyhc1 pre-mRNA in smyhc1-MO injected zebrafish embryos

A: diagram shows the gene structure of smyhc1 at the 5′-UTR. The ATG start codon, MO target site, and PCR primers are indicated. B: RT-PCR analysis of smyhc1 expression in the control and smyhc1-MO injected embryos at 24 hpf. Compared with the control, two PCR products were amplified in the smyhc1-MO injected embryos.

Fig. 3. The nuclear localization of smyhc1 pre-mRNA in smyhc1 ATG-MO injected embryos

A–D: in situ hybridization shows smyhc1 mRNA localization in the control (A, C) or smyhc1-MO (B, D) injected embryos at 24 hpf. Nuclear localization was observed in the smyhc1-MO injected embryos (B, D). E and F: nucleus localization in slow muscles revealed by immunostaining with anti-Prox1 antibody (E), together with F59 antibody (F) for slow muscles in wild type embryo at 24 hpf. G and H: in situ hybridization shows the cytosolic localization of smyhc1 mRNA in smyd1 knockdown (G) or Hsp90α1(slo) mutant (H) embryos at 24 hpf.

Fig. 4. Effects of smyhc1 knockdown on the M-line localization of myomesin-3-RFP and smyd1b-GFP in zebrafish embryos

A and B: morphological comparison of the control (A) and smyhc1-MO injected (B) embryos at 48 hpf. C and D: immunostaining with antibody (F59) shows the Smyhc1 expression and thick filament organization in the control (C) and smyhc1-MO injected (D) embryos at 24 hpf. E and F: sarcomeric localization of myomesin-3-RFP in the control (E) or smyhc1-MO injected (F) zebrafish embryos at 24 hpf. G and H: sarcomeric localization of Smyd1b-GFP in the control (G) or smyhc1-MO injected (H) zebrafish embryos at 24 hpf

Fig. 5. Effects of hsp90α1 and Unc45b knockdown on the sarcomeric localization of myomesin-3-RFP in slow muscles of zebrafish embryos

Sarcomeric localization of myomesin-3-RFP in slow muscles of myom3(mnGt0067) fish embryos (A), hsp90α1 (B) or Unc45b (C) knockdown myom3(mnGt0067) embryos, or wild type control embryos (D) at 48 hpf.

Fig. 6. Diagram shows the gene trap integration in myomesin-3 gene and PCR strategy for analyzing the expression of myomesin-3 and myomesin-3-RFP mRNA transcripts

A:. The RFP gene trap is integrated at the intron 24 of myomesin-3. A cDNA fragment covering the junction site of myomesin-3 and RFP fusion was amplified by RT-PCR. Additional sequence of 39 bp from intron 24 and 32 bp from the vector sequence upstream of RFP was found in the myomesin-3-RFP fusion transcript. myomesin-3 GenBank accession No. XM_001921030. B: RT-PCR results show the expression of myomesin-3 mRNA transcripts or myomesin-3-RFP fusion products in wild type or homozygous myom3(mnGt0067) fish embryos.

Fig. 7. The effect of RFP gene trap insertion and myomesin-3 knockdown on M-line organization in slow muscles of zebrafish embryos

A and B: morphological comparison of the control (A) and myomein-3 ATG-MO injected (B) embryos at 24 hpf. C and D: the M-line localization of myomesin-3-RFP in myom3(mnGt0067) homozygous control (C) or myomesin-3 knockdown (D) embryos at 48 hpf. E and F: negative controls of wild type embryos for GFP (E) and RFP (F) at 28 hpf and 48 hpf, respectively. G and H: sarcomeric localization of Smyd1b-GFP at the M-lines in Smyd1b-GFP heterozygous control (G) or myomesin-3 knockdown (H) zebrafish embryos at 28 hpf.

Fig. 8. The sarcomere organization in slow muscles of homozygous myomesin-3-RFP and myomesin-3 knockdown zebrafish embryos

A–C: anti-MyHC antibody (F59) staining shows the thick filament organization in slow muscles of the control (A), homozygous myom3(mnGt0067) (B), or myomesin-3 knockdown (C) embryos at 28 hpf. D–F: α-actin immunostaining shows the organization of thin filaments in slow muscles of the control (D), homozygous myom3(mnGt0067) (E), or myomesin-3 knockdown (F) embryos at 28 hpf. G–I: α-actinin immunostaining shows the organization of Z-lines in slow muscles of the control (G), homozygous myomesin-3-RFP (H), or myomesin-3 knockdown (I) embryos at 28 hpf.