Abstract 272: The Orphan Receptor GPRC5B Increases Pro-Inflammatory Signaling in Neonatal Rat Cardiac Fibroblasts and Cardiac MMP-9-expression in vivo

Background: Inflammatory processes driven by cardiac fibroblasts (CF) play a major role in cardiac fibrosis and eventually diastolic dysfunction leading to heart failure. GPRC5B, a novel G protein-coupled orphan receptor, is involved in inflammatory pathways in adipocytes and endogenously expressed in CF. Hypothesis: We assessed the impact of GPRC5B on inflammatory and fibrotic pathways in isolated neonatal rat CF and mouse heart. Methods & Results: We show that 48h-stimulation of CF with Tumor Necrosis Factor α (TNFα, 50ng/ml) or Lipopolysaccharides (LPS, 100ng/ml) leads to a significant up-regulation of GPRC5B protein levels compared to unstimulated cells (TNFα +74%, p<0.01; LPS +111%, p<0.001, n=8) . This is confirmed by evaluation of mRNA levels after 12h-stimulation ( TNFα +69%, p<0.01; LPS +61%, p<0.01, n=9) . Similarly, a mechanical stretch of 18% of CF length for 24h increases GPRC5B-mRNA by 83% (p<0.01, n=6). Adenoviral overexpression of GPRC5B results in an increased TNFα- ( +84%, p<0.01) , Interleukin 1ß- (IL1ß, +56%, p<0.05 ), Interleukin 6- (IL6, +32%, p<0.05 ) and Matrix-Metalloproteinase 9- (MMP9, +133%, p<0.01 ) mRNA production in CF ( AdLacZ vs. AdGPRC5B, n=9) . After an additional 24h-stimulation with LPS (10ng/ml), IL1ß-, IL6- and MMP9-mRNA increases as compared to AdLacZ-infected control cells ( AdLacZ+LPS vs. AdGPRC5B+LPS : IL1ß +114%, p<0.01; IL6 +113%, p<0.05; MMP9 +195%, p<0.01; n=8). Conversely, siRNA mediated knockdown to 25% of endogenous protein levels followed by 24h-stimulation with LPS (10ng/ml) lowers significantly the expression of TNFα-, IL1ß- and IL6-mRNA when compared to control-transfected cells (siNeg+LPS vs. siGPRC5B+LPS: TNFα -28%, p<0.05; IL1ß -30%, p<0.05; MMP9 -34%, p<0.05; n=8). These findings are supported by Western Blot analysis of heart tissue from GPRC5B-transgenic mice revealing an increased protein expression of MMP9 in contrast to wild type mice (WT vs. GPRC5B-TG: MMP9 +60%, p<0.001; n=10). Conclusion: GPRC5B is up-regulated in cardiac models of inflammation and mechanical stress. GPRC5B modulates cellular inflammatory response by increasing the levels of key cytokines in CF and of MMP9 expression in mouse heart pointing to a role in myocardial inflammation and cardiac remodeling.