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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

Formation of nemaline bodies and aggregates in Tg(ACTA1D286G-eGFP)high zebrafish. a Maximum projection images from time lapse of Tg(ACTA1D286G-eGFP)high fish from 56 to 71 hpf showing nemaline bodies distributed throughout the skeletal muscle (arrows). Nemaline bodies’ fragment from 59.5 hpf (arrows), coincident with the formation of aggregates at the myosepta (arrowheads). b Quantification of the mean number of nemaline bodies in Tg(ACTA1D286G-eGFP)low (n = 50 per stage) and Tg(ACTA1D286G-eGFP) (n = 48 per stage) strains. c Quantification of the percentage of fish displaying globular aggregates in Tg(ACTA1D286G-eGFP)low and Tg(ACTA1D286G-eGFP)high strains (n = 50 per stage). Error bars represent SEM from three independent experiments (n = 45 per replicate)
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Fig3: Formation of nemaline bodies and aggregates in Tg(ACTA1D286G-eGFP)high zebrafish. a Maximum projection images from time lapse of Tg(ACTA1D286G-eGFP)high fish from 56 to 71 hpf showing nemaline bodies distributed throughout the skeletal muscle (arrows). Nemaline bodies’ fragment from 59.5 hpf (arrows), coincident with the formation of aggregates at the myosepta (arrowheads). b Quantification of the mean number of nemaline bodies in Tg(ACTA1D286G-eGFP)low (n = 50 per stage) and Tg(ACTA1D286G-eGFP) (n = 48 per stage) strains. c Quantification of the percentage of fish displaying globular aggregates in Tg(ACTA1D286G-eGFP)low and Tg(ACTA1D286G-eGFP)high strains (n = 50 per stage). Error bars represent SEM from three independent experiments (n = 45 per replicate)

Mentions: To investigate the origins of the nemaline bodies within the cell, we carried out in vivo time-lapse analysis of the Tg(ACTA1D286G-eGFP)high fish. This showed, for the first time, that nemaline bodies form at the sites of muscle attachment at approximately 30 hpf and then extended into the muscle cell (Fig. 3a, Supplementary Movie S1). As development progressed, the nemaline bodies were seen to move throughout the cytoplasm (Fig. 3a, Supplementary Movie S1). The nemaline bodies were highly dynamic, rather than being tethered to the Z-disk or extracellular matrix as previously proposed [46, 57]. By 60 hpf, the characteristic rod-like bodies fragmented and disappeared from the cell, coincident with the formation of the globular aggregates at the myosepta (Fig. 3a, Supplementary Movie S2). Indeed time-lapse analysis of Tg(ACTA1D286G-eGFP)high fish showed that as nemaline bodies’ fragment, the fluorescent fusion protein previously incorporated within them can be encapsulated into globular aggregates, at the myosepta (Fig. 3a, Supplementary Movie S2).Fig. 3


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)

Formation of nemaline bodies and aggregates in Tg(ACTA1D286G-eGFP)high zebrafish. a Maximum projection images from time lapse of Tg(ACTA1D286G-eGFP)high fish from 56 to 71 hpf showing nemaline bodies distributed throughout the skeletal muscle (arrows). Nemaline bodies’ fragment from 59.5 hpf (arrows), coincident with the formation of aggregates at the myosepta (arrowheads). b Quantification of the mean number of nemaline bodies in Tg(ACTA1D286G-eGFP)low (n = 50 per stage) and Tg(ACTA1D286G-eGFP) (n = 48 per stage) strains. c Quantification of the percentage of fish displaying globular aggregates in Tg(ACTA1D286G-eGFP)low and Tg(ACTA1D286G-eGFP)high strains (n = 50 per stage). Error bars represent SEM from three independent experiments (n = 45 per replicate)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Formation of nemaline bodies and aggregates in Tg(ACTA1D286G-eGFP)high zebrafish. a Maximum projection images from time lapse of Tg(ACTA1D286G-eGFP)high fish from 56 to 71 hpf showing nemaline bodies distributed throughout the skeletal muscle (arrows). Nemaline bodies’ fragment from 59.5 hpf (arrows), coincident with the formation of aggregates at the myosepta (arrowheads). b Quantification of the mean number of nemaline bodies in Tg(ACTA1D286G-eGFP)low (n = 50 per stage) and Tg(ACTA1D286G-eGFP) (n = 48 per stage) strains. c Quantification of the percentage of fish displaying globular aggregates in Tg(ACTA1D286G-eGFP)low and Tg(ACTA1D286G-eGFP)high strains (n = 50 per stage). Error bars represent SEM from three independent experiments (n = 45 per replicate)
Mentions: To investigate the origins of the nemaline bodies within the cell, we carried out in vivo time-lapse analysis of the Tg(ACTA1D286G-eGFP)high fish. This showed, for the first time, that nemaline bodies form at the sites of muscle attachment at approximately 30 hpf and then extended into the muscle cell (Fig. 3a, Supplementary Movie S1). As development progressed, the nemaline bodies were seen to move throughout the cytoplasm (Fig. 3a, Supplementary Movie S1). The nemaline bodies were highly dynamic, rather than being tethered to the Z-disk or extracellular matrix as previously proposed [46, 57]. By 60 hpf, the characteristic rod-like bodies fragmented and disappeared from the cell, coincident with the formation of the globular aggregates at the myosepta (Fig. 3a, Supplementary Movie S2). Indeed time-lapse analysis of Tg(ACTA1D286G-eGFP)high fish showed that as nemaline bodies’ fragment, the fluorescent fusion protein previously incorporated within them can be encapsulated into globular aggregates, at the myosepta (Fig. 3a, Supplementary Movie S2).Fig. 3

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