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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

Characterization of skeletal muscle pathology in Tg(ACTA1D286G-eGFP)high zebrafish. a Skeletal muscle expressing i mosaic ACTA1D286G-eGFP, and ii overlaid with a light microscopy image of the same section showing Gomori trichrome staining, and iii enlarged. Dark regions (indicative of nemaline bodies) of disrupted muscle correspond to eGFP expression (arrows). b Correlative light and electron microscopy of Tg(ACTA1D286G-eGFP)high fish muscle at 2 dpf. bi Fluorescent image and corresponding ii electron microscopy image of skeletal muscle section containing a dense, elongated nemaline body (arrow) and enlarged in (iii). Sections are matched using nuclei positions (asterisk, plus and hash). ci Accumulations of actin filaments (arrowheads) and ii diffuse regions of filamentous actin (arrowheads), as well as ii disrupted sarcomeric regions are evident in Tg(ACTA1D286G-eGFP)high skeletal muscle, at 2 dpf unlike the iii uniform sarcomeres observed in Tg(ACTA1-eGFP) zebrafish
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Fig2: Characterization of skeletal muscle pathology in Tg(ACTA1D286G-eGFP)high zebrafish. a Skeletal muscle expressing i mosaic ACTA1D286G-eGFP, and ii overlaid with a light microscopy image of the same section showing Gomori trichrome staining, and iii enlarged. Dark regions (indicative of nemaline bodies) of disrupted muscle correspond to eGFP expression (arrows). b Correlative light and electron microscopy of Tg(ACTA1D286G-eGFP)high fish muscle at 2 dpf. bi Fluorescent image and corresponding ii electron microscopy image of skeletal muscle section containing a dense, elongated nemaline body (arrow) and enlarged in (iii). Sections are matched using nuclei positions (asterisk, plus and hash). ci Accumulations of actin filaments (arrowheads) and ii diffuse regions of filamentous actin (arrowheads), as well as ii disrupted sarcomeric regions are evident in Tg(ACTA1D286G-eGFP)high skeletal muscle, at 2 dpf unlike the iii uniform sarcomeres observed in Tg(ACTA1-eGFP) zebrafish

Mentions: Gomori trichrome staining of skeletal muscle demonstrated the presence of patches of intense purple staining, indicative of nemaline bodies, correlating with eGFP expression (Fig. 2a). Immuno-CLEM analysis revealed densely stained nemaline bodies that correlated with the eGFP-positive structures observed by confocal microscopy (Fig. 2b). Using conventional electron microscopy on Tg(ACTA1D286G-eGFP) zebrafish, we also observed filamentous actin accumulations adjacent to the sarcomere and, occasionally, the sarcomeres appeared disorganized compared to those in Tg(ACTA1-eGFP) zebrafish (Fig. 2c).Fig. 2


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)

Characterization of skeletal muscle pathology in Tg(ACTA1D286G-eGFP)high zebrafish. a Skeletal muscle expressing i mosaic ACTA1D286G-eGFP, and ii overlaid with a light microscopy image of the same section showing Gomori trichrome staining, and iii enlarged. Dark regions (indicative of nemaline bodies) of disrupted muscle correspond to eGFP expression (arrows). b Correlative light and electron microscopy of Tg(ACTA1D286G-eGFP)high fish muscle at 2 dpf. bi Fluorescent image and corresponding ii electron microscopy image of skeletal muscle section containing a dense, elongated nemaline body (arrow) and enlarged in (iii). Sections are matched using nuclei positions (asterisk, plus and hash). ci Accumulations of actin filaments (arrowheads) and ii diffuse regions of filamentous actin (arrowheads), as well as ii disrupted sarcomeric regions are evident in Tg(ACTA1D286G-eGFP)high skeletal muscle, at 2 dpf unlike the iii uniform sarcomeres observed in Tg(ACTA1-eGFP) zebrafish
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4541704&req=5

Fig2: Characterization of skeletal muscle pathology in Tg(ACTA1D286G-eGFP)high zebrafish. a Skeletal muscle expressing i mosaic ACTA1D286G-eGFP, and ii overlaid with a light microscopy image of the same section showing Gomori trichrome staining, and iii enlarged. Dark regions (indicative of nemaline bodies) of disrupted muscle correspond to eGFP expression (arrows). b Correlative light and electron microscopy of Tg(ACTA1D286G-eGFP)high fish muscle at 2 dpf. bi Fluorescent image and corresponding ii electron microscopy image of skeletal muscle section containing a dense, elongated nemaline body (arrow) and enlarged in (iii). Sections are matched using nuclei positions (asterisk, plus and hash). ci Accumulations of actin filaments (arrowheads) and ii diffuse regions of filamentous actin (arrowheads), as well as ii disrupted sarcomeric regions are evident in Tg(ACTA1D286G-eGFP)high skeletal muscle, at 2 dpf unlike the iii uniform sarcomeres observed in Tg(ACTA1-eGFP) zebrafish
Mentions: Gomori trichrome staining of skeletal muscle demonstrated the presence of patches of intense purple staining, indicative of nemaline bodies, correlating with eGFP expression (Fig. 2a). Immuno-CLEM analysis revealed densely stained nemaline bodies that correlated with the eGFP-positive structures observed by confocal microscopy (Fig. 2b). Using conventional electron microscopy on Tg(ACTA1D286G-eGFP) zebrafish, we also observed filamentous actin accumulations adjacent to the sarcomere and, occasionally, the sarcomeres appeared disorganized compared to those in Tg(ACTA1-eGFP) zebrafish (Fig. 2c).Fig. 2

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