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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

Analysis of nebulin knockdown in zebrafish muscle. a Brightfield and fluorescent images of wild-type embryos injected with two different nebulin (neb)-targeting morpholinos (MOs) compared to control uninjected embryos at 2 dpf. Successful injection is confirmed by the presence of Cascade Blue (CB) labeling. b qRT-PCR analysis showing significant knockdown of neb transcript in Neb morphants compared to control uninjected (control) at 2 dpf. Error bars represent ±SEM for three replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05. c RT-PCR analysis revealed the absence of correctly spliced, and presence of mis-spliced, neb transcript in Neb ex5 morphants compared to control uninjected (cont) and reduced expression of neb transcript in Neb ex 35 morphants at 2 dpf. β-actin was used as an amplification control. d Quantification of sarcomere lengths showed a significant decrease in neb morphants compared to controls at 2 dpf. Error bars represent ± SEM for four replicate experiments (n = 10 per experiment), *p < 0.01. e Actinin2 labeling of Z-disks in morphant and control zebrafish at 2 dpf illustrates the reduced sarcomere length in Nebulin morphants compared to controls over 10 sarcomeres (represented by the distance between the black arrow and red arrows)
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Fig7: Analysis of nebulin knockdown in zebrafish muscle. a Brightfield and fluorescent images of wild-type embryos injected with two different nebulin (neb)-targeting morpholinos (MOs) compared to control uninjected embryos at 2 dpf. Successful injection is confirmed by the presence of Cascade Blue (CB) labeling. b qRT-PCR analysis showing significant knockdown of neb transcript in Neb morphants compared to control uninjected (control) at 2 dpf. Error bars represent ±SEM for three replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05. c RT-PCR analysis revealed the absence of correctly spliced, and presence of mis-spliced, neb transcript in Neb ex5 morphants compared to control uninjected (cont) and reduced expression of neb transcript in Neb ex 35 morphants at 2 dpf. β-actin was used as an amplification control. d Quantification of sarcomere lengths showed a significant decrease in neb morphants compared to controls at 2 dpf. Error bars represent ± SEM for four replicate experiments (n = 10 per experiment), *p < 0.01. e Actinin2 labeling of Z-disks in morphant and control zebrafish at 2 dpf illustrates the reduced sarcomere length in Nebulin morphants compared to controls over 10 sarcomeres (represented by the distance between the black arrow and red arrows)

Mentions: We next examined a model of nemaline myopathy resulting from a loss of Neb to determine whether actin aggregates were a common feature associated with nemaline myopathy. We injected two neb splice-site-targeting morpholinos (both singularly and in combination) into wild-type zebrafish. The efficacy of the morpholinos was confirmed by qRT-PCR showing that neb mRNA levels are reduced by approximately 80 % by each morpholino and approximately 90 % when both morpholinos are used in combination (Fig. 7b, c). The effect on Neb was further confirmed by examining sarcomere length, with Neb morphants (Fig. 8a) displaying significantly shorter sarcomere lengths compared to controls (Fig. 7d, e).Fig. 7


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)

Analysis of nebulin knockdown in zebrafish muscle. a Brightfield and fluorescent images of wild-type embryos injected with two different nebulin (neb)-targeting morpholinos (MOs) compared to control uninjected embryos at 2 dpf. Successful injection is confirmed by the presence of Cascade Blue (CB) labeling. b qRT-PCR analysis showing significant knockdown of neb transcript in Neb morphants compared to control uninjected (control) at 2 dpf. Error bars represent ±SEM for three replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05. c RT-PCR analysis revealed the absence of correctly spliced, and presence of mis-spliced, neb transcript in Neb ex5 morphants compared to control uninjected (cont) and reduced expression of neb transcript in Neb ex 35 morphants at 2 dpf. β-actin was used as an amplification control. d Quantification of sarcomere lengths showed a significant decrease in neb morphants compared to controls at 2 dpf. Error bars represent ± SEM for four replicate experiments (n = 10 per experiment), *p < 0.01. e Actinin2 labeling of Z-disks in morphant and control zebrafish at 2 dpf illustrates the reduced sarcomere length in Nebulin morphants compared to controls over 10 sarcomeres (represented by the distance between the black arrow and red arrows)
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Related In: Results  -  Collection

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Fig7: Analysis of nebulin knockdown in zebrafish muscle. a Brightfield and fluorescent images of wild-type embryos injected with two different nebulin (neb)-targeting morpholinos (MOs) compared to control uninjected embryos at 2 dpf. Successful injection is confirmed by the presence of Cascade Blue (CB) labeling. b qRT-PCR analysis showing significant knockdown of neb transcript in Neb morphants compared to control uninjected (control) at 2 dpf. Error bars represent ±SEM for three replicate experiments with each experiment comprising a pooled samples of 20 fish, *p < 0.05. c RT-PCR analysis revealed the absence of correctly spliced, and presence of mis-spliced, neb transcript in Neb ex5 morphants compared to control uninjected (cont) and reduced expression of neb transcript in Neb ex 35 morphants at 2 dpf. β-actin was used as an amplification control. d Quantification of sarcomere lengths showed a significant decrease in neb morphants compared to controls at 2 dpf. Error bars represent ± SEM for four replicate experiments (n = 10 per experiment), *p < 0.01. e Actinin2 labeling of Z-disks in morphant and control zebrafish at 2 dpf illustrates the reduced sarcomere length in Nebulin morphants compared to controls over 10 sarcomeres (represented by the distance between the black arrow and red arrows)
Mentions: We next examined a model of nemaline myopathy resulting from a loss of Neb to determine whether actin aggregates were a common feature associated with nemaline myopathy. We injected two neb splice-site-targeting morpholinos (both singularly and in combination) into wild-type zebrafish. The efficacy of the morpholinos was confirmed by qRT-PCR showing that neb mRNA levels are reduced by approximately 80 % by each morpholino and approximately 90 % when both morpholinos are used in combination (Fig. 7b, c). The effect on Neb was further confirmed by examining sarcomere length, with Neb morphants (Fig. 8a) displaying significantly shorter sarcomere lengths compared to controls (Fig. 7d, e).Fig. 7

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