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.
View: Text | PDF
Research Article Muscle biology

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

×

Figure 4

Net39 knockdown in myoblasts causes DNA damage upon mechanical stress.

Options: View larger image (or click on image) Download as PowerPoint
Net39 knockdown in myoblasts causes DNA damage upon mechanical stress. ...
(A) Schematic of cell compression system used to induce mechanical stretch of C2C12 myoblasts. Cells are confined within pillars on glass confinement slides. (B) Immunostaining of SUN2 (red) and DAPI (blue) in C2C12 myoblasts transduced with scrambled shRNA (shscrmb) and Net39 shRNA (shNet39) at baseline and after 1 hour of stretch. Quantification of deformed nuclei at baseline and after 1 hour of stretch. Approximately 100 cells per experiment. (C) Protein levels of γH2A.X normalized to VCL loading controls in shscrmb and shNet39 myoblasts as detected by Western blot analysis. (D) Densitometry analysis of the Western blots shown in C. (E) Immunostaining of γH2A.X (red) and DAPI (blue) in shscrmb and shNet39 C2C12 myoblasts at baseline, after 1 hour of stretch, and after 1 hour of stretch plus expression of DN-KASH, which disrupts the LINC complex. (F) Quantification of γH2A.X-positive nuclei in cell-stretching experiments with shscrmb and shNet39 C2C12 myoblasts and expression of DN-KASH. Approximately 100–300 cells per experiment. (G) Immunostaining of SUN2 (green) and DAPI (blue) in shscrmb and shNet39 C2C12 myoblasts at baseline, after 1 hour of stretch, and after 1 hour of stretch plus expression of DN-KASH, which disrupts the LINC complex. (H) Quantification of deformed nuclei in cell-stretching experiments with shscrmb and shNet39 C2C12 myoblasts and expression of DN-KASH. Deformed nuclei were identified using SUN2 staining from G. Approximately 100–300 cells per experiment. Scale bars: 10 μm (B and G); 50 μm (E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are represented as mean ± SEM. Unpaired, 2-tailed Student’s t test was performed for B and D. One-way ANOVA followed by Tukey’s multiple-comparisons test was performed for F and H. n = 3 independent experiments were performed for B, D, F, and H.