Smyd1 is essential for myosin expression and sarcomere organization in craniofacial, extraocular, and cardiac muscles

Abstract

Skeletal and cardiac muscles are striated myofibers that contain highly organized sarcomeres for muscle contraction. Recent studies revealed that Smyd1, a lysine methyltransferase, plays a key role in sarcomere assembly in heart and trunk skeletal muscles. However, Smyd1 expression and function in craniofacial muscles are not known. Here, we analyze the developmental expression and function of two smyd1 paralogous genes, smyd1a and smyd1b, in craniofacial and cardiac muscles of zebrafish embryos. Our data show that loss of smyd1a (smyd1a^mb5) or smyd1b (smyd1b^sa15678) has no visible effects on myogenic commitment and expression of myod and myosin heavy-chain mRNA transcripts in craniofacial muscles. However, myosin heavy-chain protein accumulation and sarcomere organization are dramatically reduced in smyd1b^sa15678 single mutant, and almost completely diminish in smyd1a^mb5; smyd1b^sa15678 double mutant, but not in smyd1a^mb5 mutant. Similar defects are also observed in cardiac muscles of smyd1b^sa15678 mutant. Defective craniofacial and cardiac muscle formation is associated with an upregulation of hsp90α1 and unc45b mRNA expression in smyd1b^sa15678 and smyd1a^mb5; smyd1b^sa15678 mutants. Together, our studies indicate that Smyd1b, but not Smyd1a, plays a key role in myosin heavy-chain protein expression and sarcomere organization in craniofacial and cardiac muscles. Loss of smyd1b results in muscle-specific stress response.

Keywords: Cardiac muscle; Craniofacial muscle; Myosin; Sarcomere; Smyd1.

Fig. 1.

The effects of loss-of-function mutations in smyd1a and smyd1b genes on myosin heavy-chain mRNA and protein expression in myoblast craniofacial muscles. A–J: In situ hybridization shows slow myosin heavy-chain 2 (smyhc2) mRNA expression in craniofacial muscles of wildtype (WT) (A, F), smyd1a^mb5 (B, G), smyd1b^sa15678 (C, H), smyd1a^mb5; smyd1b^sa15678 double-mutant embryos (D, I), and Tg(smyd1b:Smyd1b^myc)^mb6Tg; smyd1b^sa15678 transgenic mutant (E, J) at 72 hpf. Ventral views (A–E); Side views (F–J). K–O: Immunostaining (IHC) shows the myosin heavy-chain (MHC) protein expression in craniofacial muscles of wildtype (WT) (K), smyd1a^mb5 (L), smyd1b^sa15678 (M), smyd1a^mb5; smyd1b^sa15678 (N) mutant embryos, and Tg(smyd1b:Smyd1b^myc)^mb6Tg; smyd1b^sa15678 transgenic mutant (O) at 72 hpf. The immunostaining was performed using the anti-MHC A4.1025 monoclonal antibody. Head to the left, ventral view (K–O). The craniofacial muscles including adductor mandibulae (am), intermandibularis anterior (ima), intermandibularis posterior (imp), hyohyoideus (hh), interhyoideus (ih), and sternohyoideus (sh) muscle groups are indicated by white arrows. Scale bars, 50 μm.

Fig. 2.

The effect of smyd1a and smyd1b single and double mutations on sarcomere organization in craniofacial muscles. A–L: The sarcomere organization was analyzed by immunostaining in craniofacial muscles of wildtype (WT), smyd1a^mb5 and smyd1b^sa15678 single as well as smyd1a^mb5; smyd1b^sa15678 double-mutant embryos at 96 hpf. Head to the left, ventral view. Z-line organization labeled with EA-53 anti α-actinin antibody (A7811; MilliporeSigma) (A/a–D/d). Thick filaments labeled using anti-myosin light-chain antibody (F310, DSHB) (E/e–H/h). Thin filament organization was characterized by phalloidin-TRIC staining (I/i–L/l). Part of the jaw muscles in the white boxes of picture A–L are shown at higher magnification in a–l. Scale bars, 25 μm.

Fig. 3.

Loss of smyd1b results in upregulation of hsp90α1 and unc45b gene expression in craniofacial muscles. (A–P): Whole-mount in situ hybridization was performed to determine hsp90α1 and unc45b mRNA expression in WT and mutant embryos at 48 hpf. Ventral (A–D) and side (E–H) views show hsp90α1 gene expression in craniofacial muscles of WT control (A, E), smyd1a^mb5 (B, F), smyd1b^sa15678 (C, G) or smyd1a^mb5; smyd1b^sa15678 double-mutant (D, H) embryos at 48 hpf. Ventral (I–L) and side (M–P) views show unc45b gene expression in craniofacial muscles of WT control (I, M), smyd1a^mb5 (J, N), smyd1b^sa15678 (K, O) or smyd1a^mb5; smyd1b^sa15678 double-mutant (L, P) embryos at 48 hpf. The craniofacial muscles are indicated by arrows. Scale bars, 50 μm.

Fig. 4.

The effect of smyd1a and smyd1b deficiency on myosin heavy-chain protein expression and sarcomere organization in extraocular muscles. A–H: Immunostaining (MHC-IHC) shows the myosin heavy chain protein expression in extraocular muscles of wildtype (WT) (A, E), smyd1a^mb5 (B, F), smyd1b^sa15678 (C, G) and smyd1a^mb5; smyd1b^sa15678 (D, H) mutant embryos at 72 hpf. The extraocular muscles are indicated by black arrows. The immunostaining was performed using the anti-MHC A4.1025 monoclonal antibody. Dorsal views (A–D); Ventral views (E–H). I/i–T/t: Sarcomere organization in extraocular muscles of wildtype and mutant embryos at 96 hpf. Z-lines were characterized by anti-α-actinin antibody staining (I/i–L/l). The extraocular muscles are indicated by white arrows. Thin filament organization revealed by phalloidin-TRIC staining (M/m–P/p). Thick filaments were characterized by anti-myosin light-chain antibody staining (Q/q–T/t). Part of the extraocular muscles in the white boxes of picture I–T were shown at higher magnification in i–t, respectively. Scale bars, 30 μm.

Fig. 5.

The effect of smyd1a and smyd1b single and double mutations on heart development and myosin heavy chain expression. A–E: Morphology of head and heart regions of WT control (A), smyd1a^mb5 (B), smyd1b^sa15678 (C), smyd1a^mb5; smyd1b^sa15678 double mutant (D) and Tg(smyd1b:Smyd1b^myc)^mb6Tg; smyd1b^sa15678 transgenic mutant (E) larvae at 96 hpf. Pericardial edema is indicated by arrowheads in smyd1b^sa15678 (C) and smyd1a^mb5; smyd1b^sa15678 (D) mutant larvae. F–O: Immunostaining (IHC) shows the myosin heavy-chain (MHC) protein expression in craniofacial and cardiac muscles of WT control (F, K), smyd1a^mb5 (G, L), smyd1b^sa15678 (H, M), smyd1a^mb5; smyd1b^sa15678 (I, N) and Tg(smyd1b:Smyd1b^myc)^mb6Tg; smyd1b^sa15678 transgenic mutant (J, O) embryos. The immunostaining was performed using the anti-MHC A4.1025 antibody. The arrows indicate hearts in WT and mutant larvae. Scale bars, 50 μm.

Fig. 6.

The effect of smyd1a and smyd1b single and double mutations on sarcomere organization, hsp90α1 and unc45b expression in cardiac muscle cells. A–H: Zebrafish embryos of WT control, smyd1a and smyd1b mutants were fixed at 72 hpf. The sarcomeric organization of Z-lines (A–D) and thin filaments (E–H) was analyzed by staining with anti-α-actinin antibody (EA-53) and phalloidin-TRIC, respectively. The whole heart images were shown in the button left corner of each picture. I–P: Whole-mount in situ hybridization was performed to determine hsp90α1 (I–L) and unc45b (M–P) mRNA expression in WT control (I, M), smyd1a^mb5 (J, N), smyd1b^sa15678 (K, O) or smyd1a^mb5; smyd1b^sa15678 (L, P) mutant embryos at 24 hpf. hsp90α1 and unc45b gene expression in trunk muscles and hearts are indicated by arrowheads and arrows, respectively. Scale bars, 30 μm (A–H); 150 μm (I–P).