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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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

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Figure 6

Net39 regulates Mef2c activity to protect against DNA damage induced by mechanical stress.

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Net39 regulates Mef2c activity to protect against DNA damage induced by...
(A) Upstream regulator analysis of transcription factors (TFs) for the upregulated (red) and downregulated (blue) genes from cKO myonuclei identified by snRNA-Seq. (B) Transcript levels of Mef2c mRNA detected by bulk RNA-Seq and protein levels of MEF2C and VCL loading control detected by Western blotting were measured in GP muscles from Ctrl and cKO mice. Unpaired, 2-tailed Student’s t test. n = 3 mice. (C) Top GO pathways enriched in Mef2c target genes that are upregulated in cKO myonuclei from snRNA-Seq. (D) Net39 BioID in C2C12 myotubes detects enrichment of biotinylated MEF2C, but not the myogenic transcription factors MYOD and MYOGENIN (MYOG). Total biotinylated proteins were detected using streptavidin-HRP (STV-HRP). (E) Luciferase activity was measured in the presence (+) or absence (–) of MEF2C and NET39 and was normalized to X-gal. (F) Whole-mount X-gal staining of GP muscles at P17 performed in WT and Net39-KO mice expressing the DesMef-LacZ transgene. (G) 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 overexpression of MEF2C. Experiment and analysis in Figure 4, E–H, were performed contemporaneously with G–J. (H) Quantification of γH2A.X-positive nuclei in G. (I) 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 overexpression of MEF2C. (J) Quantification of deformed nuclei using SUN2 staining from G. Scale bars: 50 μm (G); 10 μm (I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are represented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple-comparisons test and 3 independent experiments were performed for E, H, and J. n = 3 independent experiments were performed for E, H, and J.