**1. Introduction**

Skeletal muscle myogenesis, such as muscle regeneration after injury, is a biological process critical for maintaining a functional musculoskeletal system. Myogenesis generally consists of several consecutive stages, including activation of satellite cells, proliferation of myoblasts, myogenic differentiation, and fusion into multinucleated myocytes that can later become fully mature and long, differentiated muscle cells, sometimes referred to as muscle fibers [1]. This process is highly coordinated, and many factors have been shown to be involved in the regulation of myogenesis [2].

Prostaglandins (PGs) are a group of lipid mediators (LMs) playing important roles in various physiological and pharmacological processes, such as fever, inflammation, reproductive function, tissue regeneration, and myogenesis [3–6]. In skeletal muscle, PGE2 and PGF2α, are the two most important PGs. PGE2 has been shown to enhance myoblast proliferation and differentiation [4,7], and PGF2<sup>α</sup> is able to promote muscle cell survival and fusion [8,9].

PGs are derived from arachidonic acid (AA) through the activities of a series of specific enzymes. Cyclooxygenases (COXs), including COX-1 and -2, are the rate-limiting enzymes during this process. Generally, COX-1 is constitutively expressed in most cells, while COX-2 is inducible in a variety of pathological situations, such as inflammation and cancer development [10,11]. In skeletal muscle, the knowledge of COXs derives mostly from the studies of COX-2. In muscle repair or regeneration

models, COX-2 knockout mice had delayed recovery from muscle injury, suggesting that COX-2 and the downstream PGs from this pathway could be important in regenerative myogenesis, especially in the early inflammatory phase of muscle regeneration for activation of neutrophils, macrophages, and satellite cells [12,13]. However, in this model, the roles of COX-1 and -2 in myogenic processes after the inflammation phase have not been defined. Moreover, in the hind limb suspension mouse model, the induction of COX-2 is essential for muscle recovery from the atrophy caused by unloading [14].

In addition to COXs, AA is also the substrate for lipoxygenases (LOXs) [15] and cytochromes P450 (CYPs) [16]. The metabolites via these two pathways include leukotrienes and hydroxy eicosatetraenoic acids, which are biological activators of intracellular signaling [17,18]. To our knowledge, the interactions between COXs, LOXs, and CYPs have not been studied in skeletal muscle. The changes in the functionalities of COXs would cause indirect effects resulting in modified activities of LOX and/or CYPs.

In this study, we investigated the functional relevance of COX-1 and -2 in myogenesis from myoblasts to the development of multi-nucleated myotubes in both C2C12 cells and mouse primary myoblasts. Since selective inhibitors of COX could reduce the production of PG through COX-independent pathways and could be unselective under certain conditions [19], in the present studies, specific siRNAs for COX-1 and -2 were used to evaluate the effects of COX-1 and -2 on myogenic differentiation. We employed our novel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method and a AA-targeted lipidomics method package, which is able to detect 87 compounds derived from AA, 18 eicosapentaenoic acid (EPA)-derived compounds, 16 docosahexaenoic acid (DHA)-derived compounds, and 11 ethanolamides for evaluating the changes in lipid profiling after knocking down COX-1 and -2 during myogenesis. In addition, based on the morphological changes induced by siRNA treatments, a customized skeletal muscle-targeted gene array [4] was used to identify genetic components regulated by COXs and LMs. We further linked these studies with functional measurements of intracellular calcium levels in myotubes, which is an essential surrogate for a host of skeletal muscle functions. Our results demonstrate that knocking down COXs has a significant effect on the synthesis of PGs in skeletal muscle cells. However, they function in a complex LM network not limited to PGs and have significant impacts on the levels of other LMs, such as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA), which are potentially new factors released from muscle for systemic metabolic regulation. Moreover, COXs play an important role in the regulation of gene expression of contractile apparatus and Ca2<sup>+</sup> signaling, such as *Myh7*, *Cacna1s,* and *Itrp1*, which can be reflected in the changes observed in morphological and functional tests.

## **2. Results**
