Stretch-induced myoblast proliferation is dependent on the COX2 pathway

Abstract

Skeletal muscle increases in size due to weight bearing loads or passive stretch. This growth response is dependent in part upon myoblast proliferation. Although skeletal muscles are responsive to mechanical forces, the effect on myoblast proliferation remains unknown. To investigate the effects of mechanical stretch on myoblast proliferation, primary myoblasts isolated from Balb/c mice were subjected to 25% cyclical uniaxial stretch for 5 h at 0.5 Hz. Stretch stimulated myoblast proliferation by 32% and increased cell number by 41% 24 and 48 h after stretch, respectively. COX2 mRNA increased 3.5-fold immediately poststretch. Prostaglandin E2 and F_2α increased 2.4- and 1.6-fold 6 h after stretch, respectively. Because COX2 has been implicated in regulating muscle growth and regeneration, we hypothesized that stretched myoblasts may proliferate via a COX2-dependent mechanism. We employed two different models to disrupt COX2 activity: (1) treatment with a COX2-selective drug, and (2) transgenic mice null for COX2. Treating myoblasts with a COX2-specific inhibitor blocked stretch-induced proliferation. Likewise, stretched COX2^−/− myoblasts failed to proliferate compared to controls. However, supplementing stretched, COX2^−/− myoblasts with prostaglandin E2 or fluprostenol increased proliferation. These data suggest that the COX2 pathway is critical for myoblast proliferation in response to stretch.

Introduction

Skeletal muscle fibers are terminally differentiated and are incapable of undergoing mitosis to provide new myonuclei required for postnatal muscle growth [1] and hypertrophy [2]. Satellite cells, a population of quiescent muscle precursor cells that reside beneath the basal lamina, provide the predominant source of additional myonuclei for muscle growth [3], [4]. Once activated, satellite cells give rise to myoblasts that proliferate, differentiate and fuse with the growing muscle fiber [5]. Therefore, myoblast proliferation is an early cellular event critical for skeletal muscle growth.

Myoblast proliferation is governed by a multitude of signaling cascades initiated by various autocrine/paracrine growth factors and cytokines. Previous studies show that factors such as VEGF [6], IGF-1 [7] and prostaglandins [8] promote myoblast proliferation or survival in static cultures. These factors also increase in response to mechanical stress and contribute to stretch-induced proliferation in a variety of cell types. For example, mechanical stretch stimulates proliferation in endothelial cells [9] and bladder smooth muscle cells [10] by increasing VEGF expression and prostaglandin E2 (PGE2) production, respectively.

Certainly, skeletal muscle is subject to various types of mechanical stress, including fluid shear stresses [11] and eccentric and concentric contractile forces [12]. Several studies have mimicked these in vivo forces in skeletal muscle cultures to define a variety of mechano-sensitive signaling pathways in myoblasts and myotubes [13], [14]. For example, using a biaxial mechanical stretch device, Kumar et al. [15] reported increases in the number of C2C12 myoblasts and inhibited differentiation into myotubes through a Nuclear factor-kappa B (NF-κB)-dependent mechanism [15]. Furthermore, uniaxial mechanical stretch of myotubes increases protein synthesis and stimulates growth [16] through a cyclooxygenase-dependent mechanism [17].

Mechanical stretch of skeletal muscle stimulates the phospholipase A2-mediated liberation of arachidonic acid from the cell membrane [18]. Arachidonic acid is converted into the common prostaglandin precursor, PGH2, in a rate-limiting step catalyzed by cyclooxygenase. Prostaglandin synthases then convert PGH2 into a number of prostaglandins. Cyclooxygenase exists as three isoforms termed COX1, COX2 and COX3. COX1 is produced constitutively and synthesizes prostaglandins important for normal homeostasis [19]. COX3 has recently been identified as a splice variant of the COX1 gene and appears to regulate fever within the central nervous system [20]. COX2 is an immediate/early gene that is induced during cell injury, inflammation [19] and mechanical stretch [21]. COX2 has been implicated in regulating stretch-induced proliferation of smooth muscle cells [10] and retinal mesangial cells [22]. Inhibiting COX2 decreases the total number of myoblasts in regenerating skeletal muscle suggesting a potential role for COX2 in regulating myoblast proliferation [23]. Because COX2 expression increases in response to mechanical stretch in several cell types [10], [21], [22], [24] and also appears to play a role in regulating myoblast number, we hypothesized that stretched myoblasts may proliferate via a COX2-dependent mechanism.

In this report, we demonstrate that uniaxial, cyclical mechanical stretch stimulates myoblast proliferation and results in increased cell number. Furthermore, COX2 expression and activity are necessary for this stretch-induced myoblast proliferation to occur. Our observations suggest that COX2 plays a critical role in the mechanotransduction pathway(s) that regulate myoblast proliferation and provide further evidence that using COX2-selective, anti-inflammatory drugs may be detrimental to muscle growth.