Lipidomic analysis of lipid mediators derived from cyclooxygenase‐1 and ‐2 pathways reveals their new implications in skeletal muscle

Our published studies showed that treatment with pharmacological inhibitors of cyclooxygenase‐1 (COX‐1) or COX‐2 significantly inhibited myogenesis of C2C12 cells. COX‐1 and ‐2 are rate‐limiting enzymes in the synthesis of lipid mediators, including prostaglandin E 2 (PGE 2 ), PGF 2α , PGD 2 , PGI 2 , and thromboxane A 2 (TXA 2 ), from arachidonic acid (AA). They share some similarities in regulating lipid mediator production, however, previous data suggest that they seem to function differently in the dynamic process of myogenic differentiation. We systemically compared the production of lipid mediators through COX‐1 and ‐2 during myogenesis of mouse primary myoblast using lipidomic analysis to provide new mechanistic insights for their functions in skeletal muscle. Myocytes were transfected with negative control siRNA or specific siRNA against COX‐1 or ‐2 in differentiation medium (DM). 72 h later, medium was collected for lipidomic analysis, and cells were fixed to determine Fusion Index using fluorescent tagged antibody myosin heavy chain. Knockdown of COX‐1 or ‐2 significantly attenuates myogenesis of mouse primary myoblast, demonstrated a significant reduction of by fusion index from 84.8% (negative control) to 74.3% (COX‐1 siRNA) and 73.8% (COX‐2 siRNA). Compared with 0 h of transfection, after 72 h, the levels of PGE 2 , PGF 2α , and 6‐keto‐PGF 1α (PGI 2 stable derivative) in the medium increased. In contrast, during the same period, the levels of PGD 2 and TXB 2 (TXA 2 stable metabolite) decreased. Knocking down COX‐1 significantly reduced the levels of PGE 2 and PGF 2α , but did not alter the levels of 6‐keto‐PGF 1α . Knocking down COX‐2 had a similar impact on PGE 2 levels, but the effect is less pronounced than knocking down COX‐1. Even though the levels of TXB 2 and PGA 2 (PGE 2 metabolite) at 72 h are lower than at 0 h, after knocking down COX‐1, the concentrations of TXB 2 and PGA 2 significantly decreased compared with control. Moreover, COX‐1 knockdown significantly reduced the release of DHA and OEA from myocytes/myotubes into medium, but COX‐2 knockdown significantly lowered the levels of EPA in culture medium. In addition, reduced COXs activities also affect the levels of AA metabolites through lipoxygenases (LOX) pathways. 5‐ and 12/15 LOXs synthesize 5‐, 12‐, and ‐15‐HETE from AA. We found that reduced COX‐1 or ‐2 expression significantly decreased the levels of 12‐ and 15‐HETE, but no effect on 5‐HETE. In conclusion, COX‐1 could play a predominant role in the production of lipid mediators from AA during mouse primary myoblast myogenesis, and COXs could interact with LOXs to regulate production or signaling of lipid mediators. These results point to a much more complex signaling machinery arising from the regulatory functions of COX‐1 and COX‐2 with implications that can be of long‐term interest for the area of muscle myogenesis and skeletal muscle regeneration. We are now investigating these signaling pathways in animal models and in humans. Support or Funding Information NIH‐NIA P01 AG039355, the George W. and Hazel M. Jay Endowment (to MB), and the UT System STARS Program (to MB). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .