The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C₂C₁₂ myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of K_v7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and K_v7.4-bound PIP2 levels in C₂C₁₂ cells and inhibits K_v7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of K_v7.4 channels.The endocannabinoid system (ECS) refers to a large group of endogenous molecules including the two major arachidonate-derived neuromodulatory mediators, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), known as endocannabinoids (EC); several enzymes involved in the metabolism of AEA (NAPE-PLD, ABDH4, GDE1, PTPN22 for biosynthesis and FAAH for degradation) and 2-AG (DAGLα and DAGLβ for biosynthesis and MAGL, ABDH6, ABDH12, and FAAH for degradation); and two G protein-coupled receptors known as cannabinoid receptor of type-1 (CB1) and type-2 (CB2). AEA also activates the cation permeant transient receptor potential vanilloid type-1 (TRPV1) channels (1). In mammals, the ECS regulates a large number of physiological processes; alterations in its activity are in fact responsible for the onset or progression of many types of disorders affecting both the central and the peripheral nervous system as well as other organs (2–5). So far, a few studies have reported that CB1 receptor activity controls key skeletal muscle metabolic processes such as insulin signaling, glucose uptake, and fatty acid oxidation (6, 7). However, little, if anything at all, is known about the expression profile and the functional role played by the ECS during skeletal muscle development.Skeletal myogenesis is a tightly regulated process that requires coordinated changes in a large number of genes allowing proliferating myoblasts to withdraw from the cell cycle and fuse to form large multinucleated myotubes (8). Several classes of ion channels play a pivotal role in the initiation of the differentiation process. For example, the sequential activation of two distinct classes of K⁺ channels, the ether-a-go-go K_v10.1 and the inward-rectifier KIR2.1 (9, 10), is known to be one of the first molecular events that causes myoblast hyperpolarization. This event, in turn, leads to the activation of voltage-dependent T-type Ca²⁺ channels, which increase the [Ca²⁺]_i necessary to initiate myoblast commitment to differentiation into myotubes (11). More recently, members of the K_v7 (KCNQ) subfamily of voltage-activated K⁺ channels have been found to be expressed in both myoblasts and myotubes (12, 13), and, in particular, it has been shown that K_v7.4 channel expression plays a permissive role in skeletal myogenesis (14).The K_v7 subfamily comprises five subunits (K_v7.1–K_v7.5), each showing distinct tissue distribution and physiological properties. K_v7 channel function is regulated by several classes of G_q/11-coupled receptors including muscarinic (15), bradikynin (16), serotonin (17), and somatostatin receptors (18). Stimulation of these receptors leads to phospholipase C (PLC) activation and subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Thus, considering that PIP2 is strictly required for K_v7 channels activity, G_q/11-coupled receptor stimulation represents one of the most important cellular mechanisms through which this subclass of K⁺ channels is kept under negative control (19). Interestingly, the M current, which is underlied by K_v7 channels, can be also inhibited following CB1 receptor stimulation by AEA at the postsynaptic level in hippocampal neurons (20) or by stimulation of the G_q/11-coupled orphan receptor GPR55 (21).In this study, we have endeavored to understand the role played by the ECS in muscle development and its impact on K_v7 activity during myogenesis by using molecular biology, biochemical, pharmacological, morphological, and electrophysiological techniques. Our results indicate that the endocannabinoid 2-AG tonically inhibits differentiation of mouse and human myoblasts via sequential activation of CB1 receptors, reduction of PIP2 levels, and inhibition of K_v7 channel activity.     

β (CCL2) and α (CXCL10) chemokine modulations by cytokines and peroxisome proliferator-activated receptor-α agonists in Graves' ophthalmopathy

No data are present in the literature about the effect of cytokines on the prototype β chemokine (C-C motif) ligand 2 (CCL2) or of peroxisome proliferator-activated receptor α (PPARα (PPARA)) activation on CCL2 and CXCL10 chemokines secretion in fibroblasts or preadipocytes in Graves' ophthalmopathy (GO). We have tested the effect of interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα) on CCL2, and for comparison on the prototype α chemokine (C-X-C motif) ligand 10 (CXCL10), and the possible modulatory role of PPARα activation on secretion of these chemokines in normal and GO fibroblasts or preadipocytes in primary cell cultures. This study shows that IFNγ alone, or in combination with TNFα, stimulates the secretion of CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO at levels similar to those observed in controls. IFNγ and TNFα also stimulated CXCL10 chemokine secretion as expected. The presence of PPARα and PPARγ (PPARG) in primary fibroblasts or preadipocytes of patients with GO has been confirmed. PPARα activators were able to inhibit the secretion of CXCL10 and CCL2, while PPARγ activators were confirmed to be able to inhibit CXCL10 but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists. In conclusion, CCL2 and CXCL10 are modulated by IFNγ and TNFα in GO. PPARα activators inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.