Fig. 2. MYL3 zebrafish modeling and minigene assay MYL3 zebrafish modeling.
Knockdown of the zebrafish homolog of MYL3, cmlc1 causes ventricular constriction and atrial dilation that can be rescued by wild-type MYL3 but not mutant RNA harboring the patient variants c.170C>A and c.106G>T. RNA alone or plus morpholino were injected into the cytoplasm of one-cell staged embryos and allowed to develop to 48 hpf. a cmlc1 morphant embryos showed an enlarged atrium (arrow) compared with control (con) morpholino injected embryos. The cmlc1 morphant phenotype was partially rescued by coinjection of c.170C>A RNA; embryos still showed a dilated atrium (arrow), however, displayed some improvement in heart function (categorized by observed blood flow). RNA carrying the c.106G>T variant failed to rescue the cmlc1 morphant phenotype. Scale bar: 200 μm. b Fluorescent antibody stain for atrial specific MYH6 (S46, green) and ventricular enriched MYH1 (MF20, red) at 48 hpf. Note the ventricular rescue in MYL3 coinjected morphants compared with the morpholino injected embryos alone (arrow heads). Scale bar: 10 μm. c Qualitative analysis of cardiac function in embryos injected with either control or cmlc1 morpholino alone, RNA encoding MYL3, MYL3 170C>A, or MYL3 106G>T RNA alone, or coinjected with morpholino and RNA. The heart was considered functional (FH) if both chambers contracted and blood flow was observed. d Quantitative analysis of the ventricular shortening fraction (the percentage area difference between diastolic and systolic state) shows significant improvement of contractility in morphants injected with human MYL3 compared with those injected with variants c.170C>A or c.106G>T (con: 35.7±SEM 1.4 n = 10, MO: 7.3±SEM 0.8 n = 10, MO + MYL3: 17.7 ± SEM2.3 n = 7, MO + 170C>A: 9.0±SEM 1.1 n = 9, MO + 106G>T: 6.5 ± SEM0.9 n = 9. ***P ≤ 0.001, ns: not significant). Minigene assay of the MYL3 c.482-1G>A variant. The MYL3 c.482-1G>A variant results in exon 5 skipping or removal of the first nucleotide of exon 5. e Agarose gel of reverse transcription polymerase chain reaction (RT-PCR) products from a minigene assay for the MYL3 c.482-1G>A variant. The wild-type (WT) expression construct produced only products of the expected size (~300 bp), indicative of correct splicing of MYL3 exon 5. Both mutant (MUT) expression clones (generated by site-directed mutagenesis) contain a band around the expected size (~300 bp) and a dominant, smaller band (~220 bp). f Relative abundance of RT-PCR products in each lane calculated by densitometry. g Consensus splice acceptor sequences,19 MYL3 intron 4/exon 5 wild-type and mutant splice acceptor sequences. The variant (red; A) generates a new consensus splice sequence (AG, underlined) shifted one nucleotide compared with wild-type (blue; G). h The ~300-bp RT-PCR product contains a single-nucleotide deletion (G) at the start of MYL3 exon 5 (*), confirming the use of the newly generated splice acceptor sequence. The ~200 bp RT-PCR product shows complete skipping of exon 5. Based on densitometry (f), exon 5 skipping represents the more frequent event. i Amino acid alignment of wild-type myosin ELC protein sequence (Uniprot P08590), and proteins generated with exon 5 deletion (ex5_DEL) or one nucleotide deletion (1nt_DEL). Mismatches and deletions are indicated in red. Both splicing consequences would impact the EF-hand domain 2 (AA 128–163, purple) and EF-hand domain 3 (AA 163–195, green).