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Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes—MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox—to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease associated with ectopic expression of the DUX4 gene in skeletal muscle. Muscle degeneration in FSHD is accompanied by muscle tissue replacement with fat and connective tissue. Expression of DUX4 in myoblasts stimulates mesenchymal stem cells (MSC) migration via the CXCR4-CXCL12 axis. MSCs participate in adipose and connective tissue formation and can contribute to fibrosis. Here we studied the interaction between myoblasts and MSCs and the consequences of this interaction in the FSHD context. We used cell motility assays and coculture of MSCs with myoblasts to study their mutual effects on cell migration, differentiation, proliferation, and extracellular matrix formation. The growth medium conditioned by FSHD myoblasts stimulated MSCs migration 1.6-fold (p 0.04) compared to nonconditioned medium. Blocking the CXCL12-CXCR4 axis with the CXCR4 inhibitor (AMD3100) or neutralizing antibodies to CXCL12 abolished this effect. FSHD myoblasts stimulated MSC proliferation 1.5-2 times (p 0.05) compared to control myoblasts, while the presence of MSCs impaired myoblast differentiation. Under inflammatory conditions, medium conditioned by FSHD myoblasts stimulated collagen secretion by MSCs 2.2-fold as compared to the nonconditioned medium, p 0.03. FSHD myoblasts attract MSCs via the CXCL12-CXCR4 axis, stimulate MSC proliferation and collagen secretion by MSCs. Interaction between MSCs and FSHD myoblasts accounts for several important aspects of FSHD pathophysiology. The CXCL12-CXCR4 axis may serve as a potential target to improve the state of the diseased muscles.

Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.

Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases., Graphical abstract Image 1, Highlights • Double homeobox transcription factor DUX4 misregulates hundreds of genes and induces oxidative stress in human myoblasts. • ROS, notably those of mitochondrial origin, contribute to the differentiation defects in myoblasts expressing DUX4. • A subset of genes is deregulated by DUX4 indirectly, via oxidative stress. • A strategy to identify the genes deregulated by DUX4 via oxidative stress was developed. • PITX1 is deregulated by DUX4 via oxidative stress and can be targeted to improve myogenesis in DUX4-expressing myoblasts.

International audience; After fertilization, the genome is transcriptionally quiescent to allow zygote reprogramming that relies on the RNA and proteins accumulated in the oocyte and ensures the transition from the differentiated germ cells to a totipotent state. Reprogramming is followed by zygotic genome activation (ZGA). DUX4 gene encoding for a double homeobox transcription factor is one of the key ZGA drivers in humans. Its expression, essential for embryo development, is subject to precise temporal regulation and is normally observed only at early cleavage stages. DUX4 is efficiently silenced in most somatic tissues via numerous epigenetic mechanisms , while its aberrant expression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression following chromosomal rearrangements is also observed in a subset of leukemias and sarcomas; it leads to anti-cancer immune activity suppression.