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

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

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 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

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

Filamin C is a highly dynamic protein associated with fast repair of myofibrillar microdamage

Filamin c (FLNc) is a large dimeric actin-binding protein located at premyofibrils, myofibrillar Z-discs and myofibrillar attachment sites of striated muscle cells, where it is involved in mechanical stabilization, mechanosensation and intracellular signaling. Mutations in the gene encoding FLNc give rise to skeletal muscle diseases and cardiomyopathies. Here, we demonstrate by fluorescence recovery after photobleaching that a large fraction of FLNc is highly mobile in cultured neonatal mouse cardiomyocytes and in cardiac and skeletal muscles of live transgenic zebrafish embryos. Analysis of cardiomyocytes from Xirp1 and Xirp2 deficient animals indicates that both Xin actin-binding repeat-containing proteins stabilize FLNc selectively in premyofibrils. Using a novel assay to analyze myofibrillar microdamage and subsequent repair in cultured contracting cardiomyocytes by live cell imaging, we demonstrate that repair of damaged myofibrils is achieved within only 4 h, even in the absence of de novo protein synthesis. FLNc is immediately recruited to these sarcomeric lesions together with its binding partner aciculin and precedes detectable assembly of filamentous actin and recruitment of other myofibrillar proteins. These data disclose an unprecedented degree of flexibility of the almost crystalline contractile machinery and imply FLNc as a dynamic signaling hub, rather than a primarily structural protein. Our myofibrillar damage/repair model illustrates how (cardio)myocytes are kept functional in their mechanically and metabolically strained environment. Our results help to better understand the pathomechanisms and pathophysiology of early stages of FLNc-related myofibrillar myopathy and skeletal and cardiac diseases preceding pathological protein aggregation