The nuclear receptor 4A1 (nr4a1) is responsive to beta-adrenergic stress in the heart. It mediates a maladaptive response and is contributing to contractile dysfunction. A central mechanism is the regulation of GFAT-activity, the rate limiting enzyme of the Hexosamine-Biosynthesis Pathway (HBP).

The specific, nr4a1-dependent genes in cardiomyocytes are not systematically described so far. In order to distinguish the genomic interaction partners of the transcription factor (TF) Nr4a1, we performed ChIP-seq in neonatal rat myocytes (NRVMs) with GFP-tagged nr4a1-overexpression. Via a stringent bioinformatical approach, we identified 313 promoter regions with nr4a1 binding. Pathway analysis of these targets genes highlighted the role of nr4a1 in glucose-metabolism, including malate dehydrogenase 1b (MDH1B) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB-1). In Luciferase reporter assays, we were able to confirm a nr4a1-dependent activation of the MDH1b promoter via nr4a1 (fc nr4a1+reporter/reporter: 2.26, p=0.002, Mann-Whitney Test). This activation was attenuated by administration of diindolmethan (DIM), a pharmacological inhibitor of the ligand-binding domain of nr4a1.

To determine an in vivo relevance of this molecular regulation, we treated C57BL6 mice after transaortic constriction (TAC) with DIM. The animals with DIM administrations after TAC (n=5) showed by tendency a milder elevation of genes, that are related to pathological cardiac hypertrophy (nppa, nppb, myh7) compared to non-DIM treated mice (n=5). Importantly, the transcription of MDH1B was blunted in DIM-treated animals, supporting a nr4a1-dependent regulation in-vivo (p= 0.036, Mann-Whitney Test; mean±SEM sham: 1±0.3, mean±SEM TAC: 0.63±0.39 mean±SEM sham TAC, mean±SEM: 0.06±0.03 mean±SEM TAC DIM: 0,14±0.04).

Apart from regulating the HBP, nr4a1 is interacting with enzymes within the Krebs cycle in the heart and regulators of the malate metabolism. Further diet models will show a functional consequence of cardiac malate utilization in dependence of nr4a1.