HuR-dependent expression of Wisp1 is necessary for TGF??-induced cardiac myofibroblast activity

Abstract

<b>Abstract ID 25562</b> <b>Poster Board 305</b> We have previously shown that the RNA binding protein Human antigen R (HuR) directly mediates hypertrophic signaling in cardiac myocytes (CMs), and that CM-specific genetic deletion or pharmacological inhibition of HuR reduces pathological remodeling and preserves cardiac function following transverse aortic constriction (TAC)-induced pressure overload, in part through a reduction in pro-fibrotic gene expression. More recently, our lab demonstrated that HuR expression and activity is increased in isolated primary cardiac fibroblasts (CFs) in response to TGF?? treatment. Pharmacological inhibition of HuR significantly blunts the initial TGF??-dependent activation of CFs to a pro-fibrotic myofibroblast (MF) phenotype as marked by a decrease in extracellular matrix (ECM) remodeling genes, collagen gel contraction, and in&nbsp;vitro scratch closure. However, the functional role of HuR in fibroblasts during pathological cardiac remodeling in&nbsp;vivo remains unknown. To determine the functional role of HuR in CFs, we created a fibroblast-specific deletion of HuR (CF-HuR-/-) using a tamoxifen-inducible cre recombinase under control of the PDGFRα promoter. Characterization of basal cardiac structure and function of these mice shows no significant changes compared to wild-type control littermates at 10-12 weeks old following tamoxifen treatment. However, echocardiography results showpreserved cardiac function (as marked by ejection fraction) and reduced LV dilation (left ventricular end diastolic volume) in CF-HuR-/- mice at 8 weeks following TAC-induced pressure overload. In addition, we sought to determine whether HuR inhibition in mature MFs (after their initial activation) may also induce resolution of their pro-fibrotic phenotype. To do this, primary cardiac fibroblasts were treated with TGF?? for 48 hours to induce their activation to MFs followed by HuR inhibition. Results show that treatment with a HuR inhibitor for an additional 48 hours leads to a reduction of the myofibroblast marker gene Wisp1, but not periostin, regardless of whether the cells were maintained under continued TGF?? stimulation. Similarly, HuR inhibition in MFs appears to reduce the total proteomic content of the MF-derived ECM. In summary, our new data suggests that fibroblast-specific deletion of HuR reduces pathological cardiac remodeling and fibrosis following pressure overload. Furthermore, inhibition of HuR in mature myofibroblasts reduces the expression of some, but not all, myofibroblast marker genes and appears to initiate remodeling of established fibroblast-derived ECM. Our primary current funding consists of an NIH R01 (HL158671) and AHA Transformational Project Award (TPA34910086), NIH-NHLBI Training Grant T32-HL125204