Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy
Yichi Zhang, … , Ning Liu, Eric N. Olson
Yichi Zhang, … , Ning Liu, Eric N. Olson
Published July 3, 2023
Citation Information: J Clin Invest. 2023;133(13):e163333. https://doi.org/10.1172/JCI163333.
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Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy

Abstract

Mutations in genes encoding nuclear envelope proteins lead to diseases known as nuclear envelopathies, characterized by skeletal muscle and heart abnormalities, such as Emery-Dreifuss muscular dystrophy (EDMD). The tissue-specific role of the nuclear envelope in the etiology of these diseases has not been extensively explored. We previously showed that global deletion of the muscle-specific nuclear envelope protein NET39 in mice leads to neonatal lethality due to skeletal muscle dysfunction. To study the potential role of the Net39 gene in adulthood, we generated a muscle-specific conditional knockout (cKO) of Net39 in mice. cKO mice recapitulated key skeletal muscle features of EDMD, including muscle wasting, impaired muscle contractility, abnormal myonuclear morphology, and DNA damage. The loss of Net39 rendered myoblasts hypersensitive to mechanical stretch, resulting in stretch-induced DNA damage. Net39 was downregulated in a mouse model of congenital myopathy, and restoration of Net39 expression through AAV gene delivery extended life span and ameliorated muscle abnormalities. These findings establish NET39 as a direct contributor to the pathogenesis of EDMD that acts by protecting against mechanical stress and DNA damage.

Authors

Yichi Zhang, Andres Ramirez-Martinez, Kenian Chen, John R. McAnally, Chunyu Cai, Mateusz Z. Durbacz, Francesco Chemello, Zhaoning Wang, Lin Xu, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson

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Figure 3

Loss of Net39 leads to impaired nuclear envelope integrity and DNA damage.

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Loss of Net39 leads to impaired nuclear envelope integrity and DNA damag...
(A) Electron micrographs showing a Ctrl nucleus and a cKO nucleus in GP muscles at 5 months of age. (B) Immunostaining of SUN2 (green) and DAPI (blue) in Ctrl and cKO GP muscles at 5 months of age (left). Quantification of deformed nuclei. n = 3–4 mice. (C) Immunostaining of SUN2 (green), γH2A.X (red), and DAPI (blue) in Ctrl and cKO GP muscles at 5 months of age. Dashed lines outline myofiber membranes. Magnified square area highlights a Ctrl and a cKO nucleus. (D) Quantification of γH2A.X-positive nuclei (left) and the percentage of deformed and γH2A.X-positive nuclei among the total number of nuclei (right) in Ctrl and cKO GP muscles. n = 3–5 mice. (E) TUNEL staining (green), γH2A.X (red), WGA (white), and DAPI (blue) immunostaining in Ctrl and cKO GP muscles at 5 months of age. Arrowheads indicate TUNEL- and γH2A.X-positive nuclei. (F) Quantification of TUNEL-positive nuclei (left) and the percentage of TUNEL- and γH2A.X-positive nuclei over the total number of nuclei (right) in E. n = 4–5 mice and approximately 200–800 nuclei per mouse. (G) γH2A.X staining (red) of isolated Ctrl and cKO EDL muscles at 5 months of age at baseline and after 30 minutes of ex vivo stretching. Dashed lines outline the myofiber membranes. Arrowheads indicate γH2A.X-positive nuclei. (H) Quantification of γH2A.X-positive nuclei over the total number of nuclei in G. n = 4–5 mice. Scale bars: 2 μm (A); 10 μm (B and C); 50 μm (E and G). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± SEM. Unpaired, 2-tailed Student’s t test was performed for B, D, F, and H. 100 nuclei per mouse were analyzed for B, D, and H.