Abstract
Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.
Highlights
Satellite cells (SCs), the quiescent adult muscle stem cells, are responsible for muscle hypotrophy and regeneration during postnatal development and adulthood [1,2]
In order to confirm the reliability of Desmin as a marker for in-vitro-derived C2C12 myotubes, in a separate experiment we treated the same cultures with Desmin and MYH antibodies at 120 h of differentiation using two different secondary fluorescent colors and showed that the staining overlapped (Figure 1), confirming that Desmin can be used as a marker for myotube development
Our study is the first to report that E2 and eicosapentaenoic acid (EPA) induce divergent outcomes on myoblast differentiation, and that they have no synergistic effect when used in combination
Summary
Satellite cells (SCs), the quiescent adult muscle stem cells, are responsible for muscle hypotrophy and regeneration during postnatal development and adulthood [1,2]. Activation of tissue-specific transcription factors and increased gene abundance prompts the exit of dividing SCs from the cell cycle into the myogenic (myogenesis) pathway [2,3]. A reduced number of SCs and an inability to undergo myogenesis may be the outcome of, or contribute to skeletal muscle disorders such as atrophy, cachexia and sarcopenia [4]. In this regard, it has been the aim of many scientists to optimize myogenesis in-vitro to improve therapies to alleviate symptoms associated with muscle atrophy and degeneration
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