MYH 3‐associated distal arthrogryposis zebrafish model is normalized with para‐aminoblebbistatin

Abstract

Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc1−), which allowed us to compare the effects of both mutant alleles on muscle and bone development, and model the closely related disorder, spondylocarpotarsal synostosis syndrome. Heterozygous smyhc1R673H/+ embryos developed notochord kinks that progressed to scoliosis with vertebral fusions; motor deficits accompanied the disorganized and shortened slow‐twitch skeletal muscle myofibers. Increased dosage of the mutant allele in both homozygous smyhc1R673H/R673H and transheterozygous smyhc1R673H/− embryos exacerbated the notochord and muscle abnormalities, causing early lethality. Treatment of smyhc1R673H/R673H embryos with the myosin ATPase inhibitor, para‐aminoblebbistatin, which decreases actin–myosin affinity, normalized the notochord phenotype. Our zebrafish model of MYH3‐associated DA2A provides insight into pathogenic mechanisms and suggests a beneficial therapeutic role for myosin inhibitors in treating disabling contractures.

Models of Distal Arthrogryposis and Lethal Congenital Contracture Syndrome

“…To more accurately model DA2A, Whittle et al recently introduced one of the most common Freeman-Sheldon syndrome MYH3 variants, R672H, into an analogous gene in zebrafish (Danio rerio) (smyhc1 R673H ) [16]. Zebrafish breed profusely and are cost-effective compared to mice.…”

“…Autosomal dominant (AD), Autosomal recessive (AR) [9,11,12,14,. Models of human disease are rapidly becoming more sophisticated, with the ability to knock-in single nucleotide variants and create conditional (tissue specific or timedependent) knockouts [16,17]. Loss-of-function alleles, which are often easier to generate, provide critical information about gene function, but may not fully explain autosomal dominant phenotypes in which gain-of-function or dominant negative effects can cause markedly different phenotypes.…”

Anin vivoapproach to characterize novel variants associated with musculoskeletal disorders

“…The K49 and E122 residues are conserved in zebrafish TPM2 (Fig. 5A), and we recently modeled DA2A in zebrafish (Whittle et al, 2020).…”

“…Genome edited fish heterozygous for the pathogenic variant R672H in MYH3 showed musculoskeletal abnormalities consistent with joint contractures (Whittle et al, 2020). While the efficiency of genome editing technologies in zebrafish is continuing to improve, the injection of variant-encoding capped mRNAs into fertilized embryos is a well-established tool for rapidly evaluating variant pathogenicity in developing embryos and larva (Jing and Zon, 2011).…”

“…The startle response in zebrafish larva is a well-characterized reflex used to assay motor function, and we previously showed muscle function is compromised in a MYH3 model of DA using the larval startle response (Whittle et al, 2020). To understand if TPM2 variants affect muscle function in zebrafish, we injected mRNAs into one-cell stage embryos, and ran automated tracking assays in larva 6 days post-fertilization (dpf) (Fig.…”