Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways

Abstract

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE₂ and PGF_2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration.

Keywords: Cyclooxygenase; lipidomics; myogenic differentiation; skeletal muscle.

Figure 1

Verification of the high efficiency of COX-1 and COX-2 siRNA knockdown. (A) Knockdown efficiency of siRNAs targeting COX-1; (B) knockdown efficiency of siRNAs targeting COX-2; (C) COX-1 Western blot results after siRNA transfection for 48 h; (D) quantification of COX-1 Western blot results using ImageJ; (E) COX-2 Western blot results after siRNA transfection for 48 h; (F) quantification of COX-2 Western blot results using ImageJ; and (G) both COX-1 and COX-2 siRNA transfections inhibit primary myoblast myogenic differentiation. Morphological phenotypes observed after transfections with siRNAs. a: Negative control; b: COX-1 siRNA; and c: COX-2 siRNA. (H) Treatments with siRNAs significantly reduces fusion index. n = 3–4, ** p < 0.01 compared with NC.

Figure 2

COX-1 and -2 knockdown reduces the levels of key lipid mediators released by primary muscle cells. (A) Absolute quantification of lipid mediators (LMs) released in differentiation medium (DM) from primary mouse myocytes/myotubes during differentiation; (B) ratio of LMs released in DM at 72 h post-transfection comparing COX-1 siRNA or COX-2 siRNA treatment with NC transfection. n = 3, * p < 0.05 and ** p < 0.01 compared with NC; ^# p < 0.05 compared with COX-1 siRNA.

Figure 3

Knockdown of COXs reduces the levels of hydroxyeicosatetraenoic acids (HETEs) released by primary muscle cells. The levels of 12-HETE and 15-HETE, but not 5-HETE are significantly affected by the downregulation of gene expression of both COX-1 and COX-2. n = 3, * p < 0.05 and ** p < 0.01 compared with NC.

Figure 4

Treatment with PGE₂ or 15-HETE partially recovers the impaired myogenesis induced by COX-1 or -2 knockdown. Panel (A): Representative fluorescence images of morphological changes of myotubes after siRNA transfection and supplement with LMs. Blue: DAPI (4′,6-diamidino-2-phenylindole) staining; green: MHC (myosin heavy chain) staining. Panel (B): Pretreatment with PGE₂ and 15-HETE partially but significantly improved Fusion Index. n = 3, ** p < 0.01 compared with NC; ^# p < 0.05 and ^## p < 0.01 compared with COX-1 or -2 siRNA.

Figure 5

COX-1 or -2 knockdown reduces the levels of key lipid mediators released by C2C12 muscle cells. (A) Absolute quantification of LMs released in DM of C2C12; (B) ratio of LMs released in DM at 72 h post transfection comparing COX-1 siRNA or COX-2 siRNA treatment with NC transfection. n = 5, * p < 0.05 and ** p < 0.01 compared with NC; ^# p < 0.05 and ^## p < 0.01 compared with COX-1 siRNA.

Figure 6

COX-1 or -2 knockdown alters the levels of key lipid mediators in C2C12 muscle cells. n = 4, * p < 0.05 and ** p < 0.01 compared with NC; ^# p < 0.05 compared with COX-1 siRNA.

Figure 7

Knocking down COX-1 or -2 affects the expression of genes related with muscle structure and functions. (A) Genes affected by both COX-1 and COX-2 siRNA transfection; (B) genes affected by COX-1 siRNA transfection only; and (C) genes affected by COX-2 siRNA transfection only. (D) Changes in gene expression after treatment with 15-HETE for 48 h. Only genes with two-fold or greater changes, which are considered as significant changes, are listed.

Figure 8

Representative Ca²⁺ transient of mouse primary myotubes loaded with Fura-2/AM in response to 20 mM caffeine (arrows). Treatment with COX-1 siRNA induced spontaneous Ca²⁺ oscillation with reduced response to caffeine stimulation. While Ca²⁺ oscillation was not observed in myotubes treated with COX-2 siRNA, their response to caffeine stimulation was further reduced. (A) Negative control; (B) COX-1 siRNA knockdown; and (C) COX-2 siRNA knockdown.