The transcription of Nr4a1 is mediated by acute cardiac stress and activates several dependent pathways, which promote myocardial adaptation but also contributes to contractile dysfunction. Although Nr4a1 is a key metabolic regulator bearing both DNA- and ligand-binding domains, its molecular binding partners have not yet been comprehensively characterized in an unbiased manner.
Using ChIP-Seq analysis of neonatal rat ventricular myocytes (NRVMs) overexpressing GFP-tagged Nr4a1, combined with peak calling and de novo transcription factor (TF) motif analysis, we identified 313 promoter binding sites of Nr4a1. GO term analysis highlighted a role for Nr4a1 in glucose metabolism, including binding to the promoters of malate dehydrogenase 1b (Mdh1b) and succinate dehydrogenase assembly factor 1 (Sdhaf1). To verify Nr4a1-dependent regulation of these promoters, we conducted luciferase reporter assays, which showed significant transcriptional activation at both sites (fc Mdh1b Nr4a1+reporter/reporter: 1.51, p < 0.0001; Sdhaf1 Nr4a1+reporter/reporter: 1.63, p < 0.0001; Mann–Whitney test). Treatment with diindolylmethane (DIM), a pharmacological inhibitor of the Nr4a1 ligand-binding domain, attenuated this activation (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).
By performing a yeast two-hybrid screen, we searched for potential proteomic binding partners. This also revealed possible interactions of Nr4a1 with enzymes involved in glucose metabolism, such as succinate dehydrogenase (Sdhb) and phosphoglucomutase (Pgm5).
To determine the effects of Nr4a1 on cardiac glucose handling, we performed Langendorff perfusions of cardiospecific Nr4a1 knockout (KO) mice (n = 9) and controls (n = 12) using ^13C-glucose and analyzed the fractional enrichment of labeled metabolites by mass spectrometry. In KO mice, both lactic acid and pyruvate showed significantly higher enrichment than in controls (lactic acid: KO: 0.715%, WT: 0.401%; q = 0.00015; pyruvate: KO: 0.596%, WT: 0.396%; q = 0.0356; unpaired t-tests).
Furthermore, we performed animal experiments to assess the functional consequences of the cardiospecific Nr4a1 KO. As baseline experiment, we treated KO and control groups with a metabolic stressor, either malate or succinate. In controls, malate-treated mice (n = 9) exhibited significantly higher Nppb transcription than NaCl-treated animals (n = 8; p = 0.0055, Mann–Whitney test), whereas malate treatment in KO mice (NaCl: n = 8; malate: n = 7) did not alter Nppb expression. Malate administration significantly increased cardiac fibrosis in KO mice (KO-NaCl: n = 8; KO-Malate: n = 7; difference: 1.72%, p = 0.0012, Mann–Whitney test) and showed a trend toward increased fibrosis in controls (WT-NaCl: n = 8; WT-Malate: n = 9; difference: 1.05%, p = 0.11, Mann–Whitney test). Succinate-treated controls (n = 9) displayed a significantly reduced LVEF compared with NaCl-treated controls (n = 7; difference: −8.8%, p = 0.016, Mann–Whitney test), whereas succinate-treated KO mice (KO-NaCl: n = 8; KO-Succinate: n = 7) showed no decrease in LVEF (difference: 7.5%, p = 0.28, Mann–Whitney test).
Ongoing testing of protein–protein interactions and metabolomic analyses from animal experiments inducing heart failure will help us further decipher the functional influence of Nr4a1 in heart failure.
