The DUX4-HIF1α Axis in Murine and Human Muscle Cells: A Link More Complex Than Expected.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.

Hypoxia enhances human myoblast differentiation: involvement of HIF1α and impact of DUX4, the FSHD causal gene.

BACKGROUND: Hypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD). METHODS: We examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO2: 21%) or hypoxia (PO2: 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl2) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology. RESULTS: We found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation. CONCLUSION: Hypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration.

Interplay between mitochondrial reactive oxygen species, oxidative stress and hypoxic adaptation in facioscapulohumeral muscular dystrophy: Metabolic stress as potential therapeutic target.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

Hypoxia and Hypoxia-Inducible Factor Signaling in Muscular Dystrophies: Cause and Consequences.

Muscular dystrophies (MDs) are a group of inherited degenerative muscle disorders characterized by a progressive skeletal muscle wasting. Respiratory impairments and subsequent hypoxemia are encountered in a significant subgroup of patients in almost all MD forms. In response to hypoxic stress, compensatory mechanisms are activated especially through Hypoxia-Inducible Factor 1 α (HIF-1α). In healthy muscle, hypoxia and HIF-1α activation are known to affect oxidative stress balance and metabolism. Recent evidence has also highlighted HIF-1α as a regulator of myogenesis and satellite cell function. However, the impact of HIF-1α pathway modifications in MDs remains to be investigated. Multifactorial pathological mechanisms could lead to HIF-1α activation in patient skeletal muscles. In addition to the genetic defect per se, respiratory failure or blood vessel alterations could modify hypoxia response pathways. Here, we will discuss the current knowledge about the hypoxia response pathway alterations in MDs and address whether such changes could influence MD pathophysiology.

Stixilamides A and B, two new phenolic amides from the leaves of Stixis suaveolens.

The ethylacetate extracts produced from the leaves of Stixis suaveolens (Roxb.) was characterized on the basis of NMR spectra combined with extensive mass spectroscopic techniques. The chemical characterization revealed presence of two new phenolic amides which were named as stixilamides A and B.

Induction of a local muscular dystrophy using electroporation in vivo: an easy tool for screening therapeutics.

Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose-response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.

Knowledge, attitudes and practices of livestock and aquaculture producers regarding antimicrobial use and resistance in Vietnam.

The use of antibiotics in livestock production is considered a major driver of antibiotic resistance on a global scale. In Vietnam, small- and medium-scale livestock producers dominate the domestic market and regulatory pushes have done little to decrease antibiotic use. In order to inform future policy directions, this study aims to explore knowledge, attitudes, and practices amongst livestock producers to identify their perspectives on antibiotic use and resistance. A total of 392 small- and medium-scale producers specialized in pig, poultry and aquaculture production participated in the study. The results showed that the primary reason for antibiotic use reported by producers was for the treatment of infections (69%). However, prophylactic use was also evident, with farmers reporting other reasons for antibiotic use such as "animals display abnormal symptoms or behaviour" (55%), the "weather is about to change" (25%), or "animals on neighboring farms fall ill" (27%). Only one-fifth of producers demonstrated favorable attitudes towards antibiotic use and preventing antibiotic resistance. Moreover, administering antibiotics remained the preferred countermeasure directly applied by farmers at the first indication of disease (17%), compared to enacting hygiene (10%) or quarantine (5%) measures. The results showed divergent trends amongst producers, with pig producers demonstrating higher levels of knowledge, more favorable attitudes, and higher self-reported utilization of good practice. Better knowledge, attitudes, and practices were also associated with producers who engaged in efforts to explore information on antibiotic use and resistance, which improved incrementally with the number of sources consulted and hours invested. However, there were some areas where increased knowledge or more favorable attitude scores did not translate into better practices. For instance, producers with higher levels of formal education performed significantly better than those with lower education in terms of knowledge and attitude, though both groups reported similar practices. The findings of this study may support future interventions to prevent both antibiotic misuse and the development of antimicrobial resistance.

Synthesis and antiproliferativeactivity of new vinca alkaloids containing an α,ß-unsaturated aromatic side chain.

A new series of vinca-alkaloids derivatives containing various α,ß-unsaturated aromatic side chains was synthesized. Four new vinca-alkaloids derivatives showed selective cytotoxicities against KB tumor cell lines with IC50 value below 0.1 µM, thus comparable with vinblastine.