Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy

Abstract Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy

Quantification of RNA and protein levels following Actc1b morpholino knockdown.

<p>A) Representative data from western blot analysis for α-actin protein expression in <i>actc1b</i>^<i>-/-</i> and their wildtype siblings (<i>actc1b</i>^<i>+/-</i> and <i>actc1b</i>^<i>+/+</i>) at 2 dpf injected with either an Actc1b UTR, Actc1b ex2, or Standard Control MO. β-tubulin was used as a loading control. B) Quantification of western blot analysis from three independent replicate experiments of A) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007212#pgen.1007212.s005" target="_blank">S5 Fig</a>) consisting of a pooled sample of 20 tails whereby α-actin protein levels were normalized against the -tubulin loading control. Error bars represent SEM for three independent experiments, *p<0.05, **p<0.01, and #p<0.0001 using a two-way ANOVA. C) Quantitative RT-PCR analysis for zebrafish <i>acta1a</i>, <i>acta1b</i>, <i>actc1a</i>, and <i>actc1b</i> genes in tail samples from <i>actc1b</i>^<i>+/+</i>, <i>actc1b</i>^<i>+/-</i>, and <i>actc1b</i>^<i>-/-</i> at 2 dpf. Error bars represent SEM for three independent experiments each consisting of a pooled sample of 30 tail samples, *p<0.05 indicates difference from <i>actc1b</i>^<i>+/+</i> using a one-way ANOVA. D) Quantitative RT-PCR analysis for zebrafish <i>acta1a</i>, <i>acta1b</i>, <i>actc1a</i>, and <i>actc1b</i> genes in whole embryos from Actc1b ex2 and Actc1b UTR morphants compared to Standard control MO injected zebrafish at 2 dpf. Error bars represent SEM for three independent experiments each consisting of a pooled sample of 20–30 embryos, **p<0.01, and #p<0.001 indicate difference from control MO using a one-way ANOVA.</p

Characterization of phenotypic severity following Actc1a morpholino knockdown.

<p>A) <i>actc1b</i>^<i>-/-</i> and wildtype siblings (<i>actc1b</i>^<i>+/-</i> and <i>actc1b</i>^<i>+/+</i>) injected with either an Actc1a splice or Standard Control MO were stained with Actinin2 and phenotypes were scored as either wildtype, mild (small outgrowth of aggregates at the myosepta (arrowheads)) or severe (large outgrowth of aggregates at the myosepta (arrowheads) and Actinin2 positive aggregates throughout the muscle fibers (arrows)). Scale bar represents 50μm. B) Quantification of the phenotypic severity for <i>actc1b</i>^<i>-/-</i> and wildtype siblings (<i>actc1b</i>^<i>+/+</i> and <i>actc1b</i>^<i>+/-</i>) injected with Actc1a splice compared to Standard Control MO injected zebrafish. Error bars represent SEM for three independent experiments (for Actc1a MO n = 8,13,7 <i>actc1b</i>^<i>+/+</i>, n = 27,16,16 <i>actc1b</i>^<i>+/-</i> and n = 7,15,7 <i>actc1b</i>^<i>+/+</i> and for Standard Control MO n = 7,3,7 <i>actc1b</i>^<i>+/+</i>, n = 16,21,20 <i>actc1b</i>^<i>+/-</i> and n = 4,8,6 <i>actc1b</i>^<i>-/-</i>), *p<0.05 indicates a significant difference in phenotype proportions using a Chi-square test.</p

Characterization of muscle phenotypes in <i>actc1b</i>^<i>-/-</i> mutants and Actc1b morphants.

<p>A) Antibody labelling against Actinin2 and Phalloidin of <i>actc1b</i>^<i>-/-</i> mutants and wildtype siblings with Actinin2 (green) and F-actin (red) at 2 dpf and 6 dpf showing normal muscle morphology. Scale bar represents 50μm. B) Locomotion assays show a significant reduction in distance travelled by <i>actc1b</i>^<i>-/-</i> mutants compared to siblings (<i>actc1b</i>^<i>+/-</i> and <i>actc1b</i>^<i>+/+</i>) zebrafish. Error bars represent SEM for three independent experiment (n = 6,11,16 for <i>actc1b</i>^<i>+/+</i>; n = 24,23,18 for <i>actc1b</i>^<i>+/-</i>; and n = 13,9,10 for <i>actc1b</i>^<i>-/-</i> per experiment), *p<0.05 using a one-way ANOVA. C) Locomotion assays showing a significant reduction in distance travelled by Actc1b ex2 and UTR morphants compared to both Standard Control MO injected and uninjected zebrafish. No significant difference in locomotion is observed for Standard Control MO injected and uninjected zebrafish. Error bars represent median values and interquartile range (pooled samples from 3 independent experiments n = 45,48,46 for Actc1b ex2 MO; n = 45,48,33 for Actc1b UTR MO; n = 45,48,47 for Standard Control MO; and n = 48,47,45 for uninjected zebrafish), #p<0.0001 using a Kruskal-Wallis Test.</p

Quantitative RT-PCR analysis for zebrafish α-actin genes.

<p>mRNA expression of <i>acta1a</i>, <i>acta1b</i>, <i>actc1a</i>, and <i>actc1b</i> genes in the A) head (comprising the heart tissue) and B) tail (comprising predominantly skeletal muscle tissue) during zebrafish development. Error bars for A) and B) represent SEM for three independent biological replicates each consisting of a pooled sample of 20–30 embryos.</p

Characterization of phenotypic severity following Actc1b morpholino knockdown.

<p>A) <i>actc1b</i>^<i>-/-</i> and wildtype siblings (<i>actc1b</i>^<i>+/-</i> and <i>actc1b</i>^<i>+/+</i>) injected with either an Actc1b ex2, Actc1b UTR or Standard Control morpholino were stained with Actinin2 (green) and F-actin (red) and phenotypes were scored as either wildtype, mild (small outgrowth of aggregates at the myosepta (arrowheads)) or severe (large outgrowth of aggregates at the myosepta (arrowheads) and Actinin2 positive aggregates throughout the muscle fibers (arrows)). Scale bar represents 50μm. B) Quantification of phenotypic severity for <i>actc1b</i>^<i>-/-</i> and wildtype siblings injected with Actc1b ex2 and Actc1b UTR MOs compared to Standard Control MO injected zebrafish. Error bars represent SEM for three independent experiments (for Actc1b ex2 MO: n = 26,14,16 <i>actc1b</i>^<i>+/+</i>, n = 31,35,29 <i>actc1b</i>^<i>+/-</i> and n = 12,11,23 <i>actc1b</i>^<i>-/-</i>, for Actc1b UTR MO: n = 23,21,17 <i>actc1b</i>^<i>+/+</i>, n = 28,30,23 <i>actc1b</i>^<i>+/-</i> and n = 8,11,8 <i>actc1b</i>^<i>-/-</i> and for Standard Control MO: n = 11,8,10 <i>actc1b</i>^<i>+/+</i>, n = 11,13,11 <i>actc1b</i>^<i>+/-</i> and n = 9,12,4 <i>actc1b</i>^<i>-/-</i>), <i>#</i>p<0.0001 using a Chi-square test. C) Locomotion assays show a significant reduction in distance travelled by <i>actc1b</i>^<i>+/+</i> injected with an Actc1b UTR MO and Actc1b ex2 MO compared to Standard Control MO, using a Kruskal-Wallis Test. Locomotion assays show a significant reduction in distance travelled by <i>actc1b</i>^<i>+/-</i> injected with an Actc1b ex2 MO compared to Control MO, using a Kruskal-Wallis Test. No difference in distance travelled is observed between <i>actc1b</i>^<i>-/-</i> mutants injected with either an Actc1b UTR MO, Actc1b ex2 MO or Standard Control MO. Error bars represent median values with interquartile range (pooled samples from 3 independent experiments for Actc1b ex2 MO: n = 25,21,20 <i>actc1b</i>^<i>+/+</i>, n = 41,53,49 <i>actc1b</i>^<i>+/-</i> and n = 20,14,15 <i>actc1b</i>^<i>-/-</i>, for Actc1b UTR MO: n = 30,24,19 <i>actc1b</i>^<i>+/+</i>, n = 42,47,57 <i>actc1b</i>^<i>+/-</i> and n = 21,23,17 <i>actc1b</i>^<i>-/-</i> and for Standard Control MO: n = 31,28,26 <i>actc1b</i>^<i>+/+</i>, n = 41,50,52 <i>actc1b</i>^<i>+/-</i> and n = 21,14,17 <i>actc1b</i>^<i>-/-</i>). *p<0.05 and #p<0.0001.</p

Repurposing of metformin identified as a potential therapy in models of BAG3 myofibrillar myopathy

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To identify therapies we generated two zebrafish models, one conditionally expressing BAG3 P209L and one with a nonsense mutation in bag3 . Whilst transgenic BAG3 P209L expressing fish display protein aggregation, modelling the early phase of the disease, bag3 12/ 12 fish demonstrate impaired autophagic activity, exercise dependent fibre disintegration, and reduced swimming activity, consistent with later stages. Having confirmed the presence of impaired autophagy in patient samples we utilised the zebrafish model to screen a library of autophagy promoting compounds for their effectiveness at removing protein aggregates, identifying nine including Metformin. Further evaluation in our models demonstrated Metformin is not only able to remove the protein aggregates in zebrafish and human myoblasts, but is also able to rescue the fibre disintegration and swimming deficit observed in the bag3 12/ 12 fish. Therefore, repurposing Metformin provides a promising therapy for BAG3 myopathy. This study therefore illustrates an approach for the identification of treatments for rare neuromuscular diseases, using genetically matched animal models to identify mechanism, confirmation of this mechanism in patients, a targeted drug screen, and demonstration of efficacy in both an animal and human cell model, providing the necessary data for clinical translation of the findings

Genetic compensation triggered by actin mutation prevents the muscle damage caused by loss of actin protein

<div><p>The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal α-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic <i>actc1b</i> mutant exhibits mild muscle defects and is unaffected by injection of the <i>actc1b</i> targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations.</p></div