1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Cedars-Sinai Medical Center Smidt Heart Institute, Department of Cardiology Los Angeles, USA
In our previous work, we identified the nuclear receptor 4A1 (Nr4a1) as a central regulator of glucose metabolism in the heart. Nr4a1 is transcriptionally upregulated in acute cardiovascular stress situation such as stroke or acute exercise. Downstream, these metabolic alterations contribute to contractile adaption and malfunction. So far, the specific Nr4a1-dependent molecular targets in cardiomyocytes are not investigated in an unbiased manner.
In order to identify promoter binding of the transcription factor (TF) Nr4a1, we performed ChIP-seq in neonatal rat myocytes (NRVMs) with GFP-tagged Nr4a1-overexpression. Combining peak calling and DeNovo TF analysis, we identified 313 promoter regions with Nr4a1 binding. GO term analysis of these targets genes highlighted the role of Nr4a1 in glucose-metabolism, including binding at the promoter of malate dehydrogenase 1b (Mdh1b) and of succinate dehydrogenase assembling factor 1 (Sdhaf1). In Luciferase reporter assays, we were able to confirm a Nr4a1-dependent activation of the Mdh1b and Sdhaf1 promoter (fc Mdh1b Nr4a1+reporter/reporter: 1.51, p<0.0001, fc Sdhaf1 Nr4a1+reporter/reporter: 1.63, p<0.0001, Mann-Whitney Test). This activation was attenuated by administration of diindolmethan (DIM), a pharmacological inhibitor of the ligand-binding domain of Nr4a1 (fc Mdh1b Nr4a1+DIM+reporter /reporter: 1.21, p=0.004, fc Sdhaf1 Nr4a1+DIM+reporter /reporter: 1.04, p<0.0001, Mann-Whitney Test).
Looking for proteomic interactions, we performed a Yeast-Two-Hybrid Screen, which revealed enzymes related to the glucose metabolism as potential binding partners (e.g., succinate dehydrogenase (Sdhb) or Phospoglucomutase (Pgm5)). On a transcriptional level, we found a decent downregulation of Mdh1b and Sdhb in isolated cardiomyocytes from Nr4a1 KO animals (n=5) compared to control littermates (n=6) (Mdh1b: p= 0.009, Sdhb: p=0.004, Mann-Whitney Test).
To further elaborate on functional consequences of Nr4a1-dependent glucose metabolism in the heart we treated cardiomyocyte-specific Nr4a1 knockout (KO) mice with malate and succinate. Control littermates treated with malate (n=9) showed a higher transcription of Nppb (p=0.0055, Mann-Whitney Test) compared to NaCl treated mice (n=8). In Nr4a1 KO mice (NaCl: n=8, malate n=7) there was no change in Nppb transcription according to malate treatment. The malate treatment led to a significant increase of fibrosis in KO mice (KO-NaCl: n=8, KO-Malate: n=7; difference: 1.72%, p=0.0012 Mann-Whitney test) and a tendency for higher fibrosis in control littermates (WT-NaCl: n=8, WT-Malate: n=9, difference: 1.05% p=0,11, Mann-Whitney test). There was no significant change of the left ventricular ejection fraction (LVEF) between the groups (WT-NaCl: n=8, WT-Malate: n=9, p=0.24 Mann-Whitney test; KO-NaCl: n=7, KO-Malate: n=7, p=0.71, Mann-Whitney test). Succinate treated control littermates (n=9) showed a decreased LVEF compared to NaCl treated wildtype mice (n=7) (difference: -8.8%, p=0.016, Mann-Whitney test), whereas there was no decrease in LVEF in KO animals after succinate treatment (difference: 7,5% p=0.28, Mann-Whitney test).
Currently ongoing work on diet models combined with cardiac stress models will help us to decipher the functional consequences of Nr4a1-dependent glucose metabolism in heart failure.