Arachidonic Acid Impairs Primary Human Skeletal Muscle Myoblasts Proliferation and Differentiation
Authors: Geddis Alyssa , Kolb Alexander Louis 
Published: April 2020

Inflammation is a process initiated by cells in response to disease or injury. The cellular response to inflammation begins with the release of inflammatory cytokines such as TNF alpha and IL‐6. This cascade of inflammatory cytokines causes the release of arachidonic acid, a key regulator of inflammation, from the cell membrane, allowing an interaction with cyclooxygenase (COX) enzymes, mediating the synthesis of pro‐inflammatory prostaglandins (PGE2 and PGF2α). Recent studies have indicated that these inflammatory events, and particularly the release of arachidonic acid, have a major impact on how muscle cells and tissue respond to inflammation; these studies, conducted on murine C2C12 cells, demonstrated that arachidonic acid supplementation leads to increased muscle cell growth. We therefore sought to determine if similar arachidonic acid supplementation in both proliferating and differentiating primary human myoblasts leads to increased muscle cell growth. Proliferating and differentiating human myoblasts were exposed to arachidonic acid at concentrations from 50 μM‐ 0.80 μM for up to 48–72 hours respectively. Cell viability was tested using 3‐(4,5‐Dimethylthiazol‐2‐Yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay, myotube area was determined by immunocytochemistry and confocal microscopy, and anabolic signaling pathways were evaluated by Western blot and RTPCR. Our data suggests that treatment of primary human myoblasts with high concentrations of arachidonic acid (50 μM and 25 μM) leads to the release of PGE2 and PGF2α at levels higher than that of control treated cells (p<0.001 for all concentrations). High concentrations of arachidonic acid have a deleterious effect on both myoblast proliferation, myotube area, and myotube fusion. Our data demonstrates that arachidonic acid reduced proliferation by 85% and 32%, myotube area by 63% and 38% and myotube fusion index by 93% and 50% respectively (P<0.001 for all). As a potential mechanism for these decreases, we analyzed the AKT proliferation pathway and found a 90% and 37% reduction respectively in Eukaryotic translation initiation factor 4E‐binding protein 1(4EBP1) following the 48 hour arachidonic acid exposure at these concentrations (p<0.001 and p<0.05). Finally, as a potential mechanism for the change in myotube area and fusion index we analyzed the levels of myosin (specifically MYH 1,2,3,7, and 8), a protein critical for the formation of myoblasts into myotubes, by RTPCR. Our data suggests that 50 μM arachidonic acid reduced the expression of all analyzed MHY isoforms by an average of 95% (p<0.001 for all) with normal expression levels not returning until the 3.125 μM dose. Together, our data demonstrates that high doses of arachidonic acid reduce primary human myoblast differentiation. These findings suggests that the response of primary human myoblasts are different than murine C2C12 cells supplemented with high doses of arachidonic acid.

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