Prostaglandin E2 is a Negative Regulator of Fibroadipogenic Progenitor Differentiation in Traumatically Denervated Skeletal Muscle

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.