Hypoxia promotes proliferation and inhibits myogenesis in broiler satellite cells.

Breast muscle myopathies in broilers compromise meat quality and continue to plague the poultry industry. Broiler breast muscle myopathies are characterized by impaired satellite cell (SC)-mediated repair, and localized tissue hypoxia and dysregulation of oxygen homeostasis have been implicated as contributing factors. The present study was designed to test the hypothesis that hypoxia disrupts the ability of SC to differentiate and form myotubes, both of which are key components of myofiber repair, and to determine the extent to which effects are reversed by restoration of oxygen tension. Primary SC were isolated from pectoralis major of young (5 d) Cobb 700 chicks and maintained in growth conditions or induced to differentiate under normoxic (20% O2) or hypoxic (1% O2) conditions for up to 48 h. Hypoxia enhanced SC proliferation while inhibiting myogenic potential, with decreased fusion index and suppressed myotube formation. Reoxygenation after hypoxia partially reversed effects on both proliferation and myogenesis. Western blotting showed that hypoxia diminished myogenin expression, activated AMPK, upregulated proliferation markers, and increased molecular signaling of cellular stress. Hypoxia also promoted accumulation of lipid droplets in myotubes. Targeted RNAseq identified numerous differentially expressed genes across differentiation under hypoxia, including several genes that have been associated with myopathies in vivo. Altogether, these data demonstrate localized hypoxia may influence SC behavior in ways that disrupt muscle repair and promote the formation of myopathies in broilers.

The Myotube Analyzer: how to assess myogenic features in muscle stem cells.

BACKGROUND: The analysis of in vitro cultures of human adult muscle stem cells obtained from biopsies delineates the potential of skeletal muscles and may help to understand altered muscle morphology in patients. In these analyses, the fusion index is a commonly used quantitative metric to assess the myogenic potency of the muscle stem cells. Since the fusion index only partly describes myogenic potency, we developed the Myotube Analyzer tool, which combines the definition of the fusion index with extra features of myonuclei and myotubes obtained from satellite cell cultures. RESULTS: The software contains image adjustment and mask editing functions for preprocessing and semi-automatic segmentation, while other functions can be used to determine the features of nuclei and myotubes. The fusion index and a set of five novel parameters were tested for reliability and validity in a comparison between satellite cell cultures from children with cerebral palsy and typically developing children. These novel parameters quantified extra nucleus and myotube properties and can be used to describe nucleus clustering and myotube shape. Two analyzers who were trained in cell culture defined all parameters using the Myotube Analyzer app. Out of the six parameters, five had good reliability reflected by good intra-class correlation coefficients (> 0.75). Children with cerebral palsy were significantly different from the typically developing children (p < 0.05) for five parameters, and for three of the six parameters, these differences exceeded the minimal detectable differences. CONCLUSIONS: The Myotube Analyzer can be used for the analysis of fixed differentiated myoblast cultures with nuclear and MyHC staining. The app can calculate the fusion index, an already existing parameter, but also provides multiple new parameters to comprehensively describe myogenic potential in its output. The raw data used to determine these parameters are also available in the output. The parameters calculated by the tool can be used to detect differences between cultures from children with cerebral palsy and typically developing children. Since the program is open source, users can customize it to fit their own analysis requirements.