Abstract P314: TGFβ2-Mediated Cardiac Fibroblast Activation Is Dependent On BET Bromodomain Proteins

Abstract

Heart failure is a common and morbid disease frequently caused by dilated cardiomyopathy (DCM). Fibrotic remodeling is a key feature of DCM. Stress signaling pathways trigger transdifferentiation of quiescent cardiac fibroblasts into myofibroblasts (MF). Bromodomain and extra-terminal (BET) epigenetic reader proteins are stress-responsive distal signaling integrators that drive fibrosis. Here, we explored the role of BETs on distinct inflammatory pathways in ex vivo cultured cardiac fibroblasts. The Langendorff hanging heart procedure was used to isolate and culture primary non-myocytes from wild type hearts, which are almost exclusively fibroblasts. Cells were stimulated with TGFβ2 to trigger MF transdifferentiation. Prior to TGFβ2 treatment, fibroblasts were treated with 1 of the following: vehicle, the BET inhibitor JQ1, or siRNA targeting either Smad3 or Rela . MF-specific gene expression was assessed by Q-PCR. α-smooth muscle actin (αSMA) levels were assessed by immunocytochemistry (ICC) and immunoblotting. NFκB transcriptional activity was evaluated using a dual luciferase reporter assay. TGFβ2 activated MF-specific gene expression ( Acta2 , 9.0-fold; Col3a1 , 3.6-fold; Ctgf, 10.1-fold; Postn , 12.8-fold; p<0.05 for all vs. vehicle), and increased αSMA levels (1.5-fold by ICC and immunoblotting; p<0.05 vs. vehicle) in cultured primary cardiac fibroblasts. This was ablated by JQ1 and Smad3 siRNA. By contrast, Rela siRNA alone induced MF-specific gene expression ( Acta2 , 6.9-fold; Col3a1 , 7.0-fold; Ctgf, 3.7-fold; Postn , 11.2-fold; p<0.05 for all vs. vehicle), and increased αSMA staining by ICC (3.4-fold; p<0.05) but not protein (1.3-fold; p=NS). TGFβ2 further potentiated MF-specific gene expression ( Acta2 , 17.1-fold; Col3a1 , 7.1-fold; Ctgf, 13.2-fold; Postn , 42.5-fold; p<0.05 for all vs. vehicle) and αSMA protein (1.7-fold; p<0.05) in fibroblasts treated with Rela siRNA. NFκB transcriptional activity was reduced by TGFβ2 (-2.9-fold; p<0.05) and with JQ1 pre-treatment (-2.7-fold; p<0.05). These data define a critical role for BETs in TGFβ2-mediated gene transcription in cardiac fibroblasts. Surprisingly, NFκB does not appear to be a key activator of TGFβ2-stimulated fibroblast transdifferentiation.