fla encodes zebrafish SMYD1. (A) Genetic and physical map of the zebrafish fla region. A point mutation was identified in exon 2 of the smyd1 gene. In the chromatograms, the fla nonsense mutation in codon 73 of smyd1 is indicated with an arrowhead. Morpholino knockdown of smyd1 phenocopies, whereas ectopic expression of wild-type (wt) SMYD1 suppresses, the fla mutant phenotypes. (B–E) Ectopic expression of wt smyd1b mRNA (smyd1b^wt) can rescue the heart and skeletal muscle phenotype of 70% of fla mutants, whereas injection of fla mutant mRNA (smyd1b^fla) has no effect. (E) Ultrastructural architecture of fast-twitch skeletal muscle cells is restored by the ectopic expression of wt smyd1b RNA. (F–I) Inhibition of smyd1 function by morpholino injection phenocopies the fla heart and skeletal muscle phenotype. (F–H) 97% of wt embryos injected with MO1-zsmyd1 or MO2-zsmyd1 (G) are indistinguishable from fla mutants (F) and display lack of cardiac contractility and impairment of skeletal muscle function, whereas injection of corresponding 5-bp mispair morpholinos do not evoke a heart and skeletal muscle phenotype (H). (I) Electron microscopy analysis of smyd1 injected with MO1-zsmyd1 reveals disruption of myofibrillar organization in fast-twitch skeletal muscle cells. All error bars represent s.d. skm, skeletal muscle.

SMYD1 is expressed in the heart and fast-twitch skeletal muscle cells, and interacts with myosin at the sarcomeric M-line. (A,B) smyd1 RNA is strongly expressed in somitic muscle cells (A) and both heart chambers (B). (C–E) smyd1-specific in situ hybridization, in combination with an immunostain of slow-twitch skeletal muscle fibers (F59, green) of transversal sections through the somitic muscles of a wild-type (wt) embryo. smyd1 is expressed in fast-twitch and is absent in slow-twitch skeletal muscle cells. (F–H) Immunohistochemistry of murine heart sections reveals that SMYD1 protein (green) localizes to the sarcomere and the nucleus of cardiomyocytes. Cell nuclei are counterstained with DAPI (blue). (I,J) Co-immunostains of SMYD1 and α-Actinin (I), as well as SMYD1 and myomesin (J), in murine heart sections reveal localization of SMYD1 to sarcomeric M-lines. (K) The scheme outlines different SMYD1b deletion variants that are used for the myosin in vitro pulldown assay (turquoise, pre-SET and post-SET domain; blue, MYND domain; purple, C-terminus). SMYD1b^wt–GST (lane 1) and SMYD1b^391del–GST (lane 4) proteins interact with myosin from rabbit muscle in an in vitro pulldown assay, whereas SMYD1b^89del -GST (lane 2) and SMYD1b^278del–GST (lane 3) do not bind myosin. GST (lane 5) and UNC-45b (lane 6) are used as negative and positive myosin-binding controls, respectively.

SMYD1a–GFP associates with nascent myosin. (A–E) Co-injection of low levels of UNC-45b morpholinos, as a model of decelerated myofibrillogenesis, and smyd1a-gfp plasmids. (A) At 48 hpf, SMYD1a–GFP first colocalizes with myosin in the sarcomeric A-band. (B) At 72 hpf, when myofibril assembly is completed, SMYD1a–GFP shuttles to the M-line. (C–E) Immunostains with α-Actinin-specific antibodies reveal striation, confirming relocalization of SMYD1a–GFP to sarcomeric M-lines.

Increased RNA levels of thick filament chaperones unc-45b and hsp90a1 in fla heart and fast-twitch skeletal muscle cells. (A) mRNA levels of sarcomeric constituents are not affected by the fla mutation. In situ hybridizations of thick- (cmlc2, vmhc, amhc), thin- (tpm4, tnnt2) and elastic-filament (titin) transcripts in fla mutants at 48 hpf indicate that their transcription is not affected by the loss of SMYD1 function. By contrast, unc-45b (B–D) and hsp90a1 (E–G) are strongly upregulated in cardiomyocytes and fast-twitch skeletal muscle cells of fla compared with that in wild-type (wt) embryos at 48 hpf, as revealed by in situ hybridization and qRT-PCR. (H–K) Muscle chaperone hsp90a1 is only upregulated in fla (I; V, ventricle, A, atrium) but not in other zebrafish mutants with disturbed sarcomeric units, such as silent heart (sih) (J) and tell tale heart (tel) (K). All error bars represent s.d.

Hsp90a1, UNC-45b and SMYD1 are co-regulated during sarcomerogenesis. (A–D) Expression of smyd1 is elevated in the thick filament mutants steif (B) and akineto (D) at 48 hpf. (E,F) smyd1 mRNA levels are also increased in fla embryos. (G) As shown by western blotting, protein levels of distinct sarcomeric myosins, such as atrial and ventricular meromyosin (MF20), atrial-specific myosin heavy chain (S46) and fast-twitch skeletal muscle myosin (EB165) are substantially reduced in fla at 48 hpf. β-actin is used as control.