Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

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.

Regulation of Skeletal Muscle Satellite Cell Differentiation by Omega-3 Polyunsaturated Fatty Acids: A Critical Review

“…Myoblast proliferation and gene/protein expression of MyoG were reduced at both 48 and 72 h with EPA and DHA treatment ( Zhang et al, 2019 ). In a similar study, using EPA only at the same concentration (50 μM) for 48 h resulted in significantly fewer myotubes, lower fusion index, and reduced expression of MyoD , MyoG , and Tmem8c (myoblast fusion related gene) compared to the control vehicle ( Lacham-Kaplan et al, 2020 ). However, increased expression of Myf6 ( Mrf4 ) and Myf5 was observed.…”

“…Overall, the effects of EPA and DHA on the formation of myotube and transcriptional regulation of myogenic differentiation are inconsistent ( Figure 3 ). Three out of the six studies reported a decrease in myotube formation and expression of genes involved in myogenic differentiation ( Myf5 , MyoD , MyoG , and MRF4 ; Hsueh et al, 2018 ; Zhang et al, 2019 ; Lacham-Kaplan et al, 2020 ). On the contrary, three studies ( Saini et al, 2017 ; Shin et al, 2017 ; Xu et al, 2018 ) reported an increase in myotube formation and expression of genes involved in myogenic differentiation with EPA or DHA treatment.…”

“…Studies that differentiate myoblasts for 3-6 days and subsequently treat myotubes with EPA and/or DHA for 24 or 48 h aim to understand terminal maturation and fusion of the myotube. Most study designs include treatment of skeletal muscle cells with both EPA and DHA and few studies treated with EPA or DHA alone (Saini et al, 2017;Løvsletten et al, 2018;Kim et al, 2019;Lacham-Kaplan et al, 2020). Under normal conditions, proliferation and differentiation of SCs occur in response to injury and inflammation.…”

“…One other source of variation in the results obtained by these studies could be the duration of treatment of the C2C12 cells with EPA or DHA. In some studies, the EPA and DHA were added to the differentiation media and present through the period of differentiation days ( Saini et al, 2017 ; Hsueh et al, 2018 ; Xu et al, 2018 ; Lacham-Kaplan et al, 2020 ). In other studies, the cells were differentiated for 3–7 days and then treated with EPA or DHA for 16, 24, or 48 h before investigating myotube formation and expression of genes involved in myogenesis.…”

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function

“…Estrogen amplifies the effects of retinoids on ameliorating obesity in female mice ( 55 ). Estrogen enhances muscle mass and strength in vivo and promotes muscle regeneration ( 56 , 57 , 58 ). Notably, estrogen action in muscle increases insulin sensitivity, prevents lipid accumulation, and promotes metabolic health ( 59 ).…”