ABSTRACT
The G-protein-coupled receptor kinase 2 (GRK2) plays a major role in cardiovascular diseases, and its expression is increased in heart failure. However, only little is known about factors being involved in up-regulation of GRK2 expression through transcriptional regulation of its promoter. Since the transcription factor early-growth response 1 (EGR-1) is also up-regulated in patients with heart failure, we tested the hypothesis that EGR-1 regulates GRK2 transcription. Stimulation of immortalized rat cardiomyocytes (H9c2) with phorbol 12-myristate 13-acetate (PMA) resulted in up-regulation of Egr-1 and subsequently of Grk2 mRNA expression, with maximum Grk2 expression (p = 0.008) 5 hr after PMA stimulation and being abolished by actinomycin D, indicating a transcriptional mechanism. To identify naturally occurring variants affecting promoter transcriptional activity, we identified a novel G(-43)A polymorphism (rs182084609), which surrounded a putative EGR-1-binding site. While the minor A allele frequency was rare (0.02), this variant was used to explore regulation by EGR-1 and promoter construct with altered alleles at nt-43 were subjected of reporter assays in human embryonic kidney cells (Hek293). Here, EGR-1 over-expression resulted in a more than twofold increase in GRK2 promoter activity but only in the presence of the G-allele (p = 0.04). In electrophoretic mobility shift assays, EGR-1 over-expression resulted in a specific binding of transcription factors only to the G oligonucleotide. Finally, EGR-1 over-expression resulted in increased GRK2 mRNA expression (p = 0.03). We identified EGR-1 as a regulator of GRK2 transcription. Suppression of GRK2 expression by inhibition of EGR-1 binding to GRK2 might be a promising approach to mitigate adrenergic desensitization.
Subject(s)
Early Growth Response Protein 1/metabolism , G-Protein-Coupled Receptor Kinase 2/metabolism , Cloning, Molecular , Computer Simulation , Early Growth Response Protein 1/genetics , Electrophoretic Mobility Shift Assay , Female , G-Protein-Coupled Receptor Kinase 2/genetics , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Promoter Regions, Genetic , Sequence Analysis, DNA , Tetradecanoylphorbol Acetate/pharmacology , Up-Regulation/drug effectsABSTRACT
OBJECTIVES: Coronary artery disease (CAD)-associated ischemic heart failure is characterized by dysregulated gene expression which is partly mediated by microRNAs (miRNAs). While the muscle-specific miR-1 and miR-133 are involved in cardiac development and hypertrophy, their role in heart failure resulting from CAD is unknown. We, therefore, tested the hypothesis that cardiac miR-1 and miR-133 expression is associated with signs of heart failure in patients undergoing coronary artery bypass grafting. METHODS: 83 patients were included in this prospective study. Cardiac index and vascular pressures were measured under general anesthesia and the miRNA expression was quantified (RNase protection assay and real-time PCR) from samples of the right atrial myocardium. RESULTS: miR-133 expression decreased significantly with increased severity of heart failure, as indicated by a greater New York Heart Association (NYHA) functional class (p = 0.014) and increased pulmonary artery occlusion pressure (p = 0.045). Furthermore, patients with NT-proBNP concentrations >1,800 pg/ml showed a 25% decrease in miR-133 expression compared to patients with concentrations <300 pg/ml (p = 0.023). In contrast, no associations were detected for miR-1 expression. CONCLUSIONS: In surgical CAD patients, a decreased miR-133 expression is associated with variables characteristic of heart failure. This supports a role for miR-133 but not miR-1 in the adaption to and/or remodeling of the ischemic heart.
