Abstract P346: Regulation of GRK5 in Myocytes Is Mediated by Nuclear Transcription Factor [ê]B: A New Clue in Heart Disease

Abstract

G protein coupled receptor kinase-5 (GRK5) plays a key role in the heart and it’s expression is increased in heart failure. Moreover, a human mutation within its amino-terminus appears to play a role in therapeutic responses to β-blockers in heart failure patients. Nuclear transcription factor kappa B (NF-κB), a ubiquitous transcription factor, is involved in the regulation of numerous genes in various tissues and activation of NF-κB has also been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the GRK5 gene and GRK5 expression in neonatal rat ventricular myocytes (NRVMs). In analyzing the 5’-flanking DNA of GRK5 , the presence of a potential NF-κB binding site was observed in the promoter. Phorbol myristate acetate (PMA), a known stimulator of NF-κB, increased the levels of GRK5 in NRVMs. Conversely, treatment of NRVMs with N-acetyl cysteine (NAC), a known inhibitor of NF-κB, decreased the levels of GRK5 compared to control cells. Utilizing electrophoretic mobility shift assay (EMSA) with nuclear extracts from NRVMs treated with or without PMA, it was found that proteins bound to the GRK5 promoter containing the putative NF-κB binding site. When an antibody specific for NF-κB was utilized, a super-shift of the proteins binding to the putative NF-κB binding site was observed. Furthermore, interaction of NF-κB with GRK5 was confirmed by shift Western blot. Chromatin immunoprecipitation (ChIP) showed dynamic recruitment of both p50 and p65 to the NF-κB site of GRK5 promoter after treatment of NRVMs with PMA. Treatment of myocytes with siRNA and subsequent knock-down of NF-κB p65 decreased the levels of GRK5 as judged by real time quantitative PCR. Finally, adenovirus mediated overexpression of a dominant negative IκB-α in NRVMs inhibited the levels of GRK5. Taken together, our present studies suggest that NF-κB plays a potential role in the regulation of GRK5 transcription in myocytes and this may translate to key expressional changes seen in heart disease.