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Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Sztal TE, Zhao M, Williams C, Oorschot V, Parslow AC, Giousoh A, Yuen M, Hall TE, Costin A, Ramm G, Bird PI, Busch-Nentwich EM, Stemple DL, Currie PD, Cooper ST, Laing NG, Nowak KJ, Bryson-Richardson RJ - Acta Neuropathol. (2015)

Bottom Line: Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

ABSTRACT
Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

No MeSH data available.


Related in: MedlinePlus

Quantification of muscle function in Tg(ACTA1-eGFP) zebrafish. a Quantification of the maximum acceleration recorded from touch-evoked response assays of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish compared to control zebrafish at 2 dpf. Error bars represent SD for n = 15–19 zebrafish, *p < 0.05. b qRT-PCR analysis of ACTA1-eGFP expression in transgenic lines at 2 dpf. No significant difference was observed between Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish. Error bars represent ±SEM for four replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05, **p < 0.01. c, d Quantification of the c number of small movements and d distance traveled by Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high and Tg(ACTA1D286G-eGFP)low and Tg(ACTA1wildtype-eGFP)low strains compared to control fish at 6 dpf. Error bars represent ±SEM for three replicate experiments (n = 48 per experiment), *p < 0.05, **p < 0.01
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Fig4: Quantification of muscle function in Tg(ACTA1-eGFP) zebrafish. a Quantification of the maximum acceleration recorded from touch-evoked response assays of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish compared to control zebrafish at 2 dpf. Error bars represent SD for n = 15–19 zebrafish, *p < 0.05. b qRT-PCR analysis of ACTA1-eGFP expression in transgenic lines at 2 dpf. No significant difference was observed between Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish. Error bars represent ±SEM for four replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05, **p < 0.01. c, d Quantification of the c number of small movements and d distance traveled by Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high and Tg(ACTA1D286G-eGFP)low and Tg(ACTA1wildtype-eGFP)low strains compared to control fish at 6 dpf. Error bars represent ±SEM for three replicate experiments (n = 48 per experiment), *p < 0.05, **p < 0.01

Mentions: In addition to the Tg(ACTA1D286G-eGFP)high zebrafish strain that showed high ACTA1D286G-eGFP expression, we also constructed a Tg(ACTA1D286G-eGFP) low-expressing strain (Tg(ACTA1D286G-eGFP)low; Fig. 4b). We analyzed the frequency of nemaline bodies during early larval stages in both transgenic strains. We observed almost no nemaline bodies in the Tg(ACTA1D286G-eGFP)low strain correlating with the much lower level of ACTA1D286G-eGFP expression (Figs. 3b, 4b). Contrastingly, the Tg(ACTA1D286G-eGFP)high strain showed the presence of nemaline bodies at 2 dpf, followed by a dramatic reduction at 4 dpf (Fig. 3b). Quantification of the aggregate phenotype identified an increase in the percentage of fish developing globular aggregates in skeletal muscle at 4 dpf, coincident with the disappearance of nemaline bodies (Fig. 3c). This supports the suggestion that early forming nemaline bodies may be one source of the ACTA1 that forms the globular aggregates.Fig. 4


Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Sztal TE, Zhao M, Williams C, Oorschot V, Parslow AC, Giousoh A, Yuen M, Hall TE, Costin A, Ramm G, Bird PI, Busch-Nentwich EM, Stemple DL, Currie PD, Cooper ST, Laing NG, Nowak KJ, Bryson-Richardson RJ - Acta Neuropathol. (2015)

Quantification of muscle function in Tg(ACTA1-eGFP) zebrafish. a Quantification of the maximum acceleration recorded from touch-evoked response assays of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish compared to control zebrafish at 2 dpf. Error bars represent SD for n = 15–19 zebrafish, *p < 0.05. b qRT-PCR analysis of ACTA1-eGFP expression in transgenic lines at 2 dpf. No significant difference was observed between Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish. Error bars represent ±SEM for four replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05, **p < 0.01. c, d Quantification of the c number of small movements and d distance traveled by Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high and Tg(ACTA1D286G-eGFP)low and Tg(ACTA1wildtype-eGFP)low strains compared to control fish at 6 dpf. Error bars represent ±SEM for three replicate experiments (n = 48 per experiment), *p < 0.05, **p < 0.01
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Related In: Results  -  Collection

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Fig4: Quantification of muscle function in Tg(ACTA1-eGFP) zebrafish. a Quantification of the maximum acceleration recorded from touch-evoked response assays of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish compared to control zebrafish at 2 dpf. Error bars represent SD for n = 15–19 zebrafish, *p < 0.05. b qRT-PCR analysis of ACTA1-eGFP expression in transgenic lines at 2 dpf. No significant difference was observed between Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high zebrafish. Error bars represent ±SEM for four replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05, **p < 0.01. c, d Quantification of the c number of small movements and d distance traveled by Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high and Tg(ACTA1D286G-eGFP)low and Tg(ACTA1wildtype-eGFP)low strains compared to control fish at 6 dpf. Error bars represent ±SEM for three replicate experiments (n = 48 per experiment), *p < 0.05, **p < 0.01
Mentions: In addition to the Tg(ACTA1D286G-eGFP)high zebrafish strain that showed high ACTA1D286G-eGFP expression, we also constructed a Tg(ACTA1D286G-eGFP) low-expressing strain (Tg(ACTA1D286G-eGFP)low; Fig. 4b). We analyzed the frequency of nemaline bodies during early larval stages in both transgenic strains. We observed almost no nemaline bodies in the Tg(ACTA1D286G-eGFP)low strain correlating with the much lower level of ACTA1D286G-eGFP expression (Figs. 3b, 4b). Contrastingly, the Tg(ACTA1D286G-eGFP)high strain showed the presence of nemaline bodies at 2 dpf, followed by a dramatic reduction at 4 dpf (Fig. 3b). Quantification of the aggregate phenotype identified an increase in the percentage of fish developing globular aggregates in skeletal muscle at 4 dpf, coincident with the disappearance of nemaline bodies (Fig. 3c). This supports the suggestion that early forming nemaline bodies may be one source of the ACTA1 that forms the globular aggregates.Fig. 4

Bottom Line: Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

ABSTRACT
Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

No MeSH data available.


Related in: MedlinePlus