A cardiac superenhancer regulates metabolic flexibility via retinoid acid signaling

Daniel Finke (Heidelberg)1, V. Sunder (Heidelberg)1, L. Schanze (Heidelberg)1, L. Sauerbrey (Heidelberg)1, K. Misura (Heidelberg)1, M. Heckmann (Heidelberg)1, T. Meßmer (Heidelberg)1, B. Meder (Heidelberg)1, H.-J. Gröne (Heidelberg)2, H. A. Katus (Heidelberg)1, N. Frey (Heidelberg)1, L. H. Lehmann (Heidelberg)1

1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Deutsches Krebsforschungszentrum (DKFZ) Heidelberg, Deutschland


The individual response to environmental stress is thought to be reflected and memorized in epigenetic modifications. We hypothesized that enhancer activation might be an early event after transient metabolic stress with maintained susceptibility for cardiovascular diseases.

We used High Fat Diet (HFD) in C57BL6 mice for 10 weeks to induce obesity in comparison to transient overweight with dietary fastening (HFD for 5 weeks and Low Fat Diet (LFD) for 5 weeks). Based on bodyweight, the reverse feeding group completely recovered from overweight to the level of control LFD feeding (LFD (n=15): 27.76g ± 0.37g, HFD (n=15): 32.93g ± 0.59g, Reverse (n=15): 28.01g ± 0.67g; mean ± SEM). Using ChIP-seq from isolated cardiomyocytes, we found 21.934 H3K4me1-positive enhancer and 330 differentially regulated H3K27ac-positive enhancers after HFD (FDR < 0.1). Particularly one superenhancer (SE), containing one coding region (Inhibitor of DNA-Binding 1, Id1) and two H3K4me1-positive enhancers (metabolic enhancer (ME) 1 and 2) was persistently activated despite reverse feeding. To further investigate the biological consequences of the metabolic SE, we generated three cardiomyocyte-specific knockout mouse lines with the use of the CRISPR/Cas9 system (Id1-cKO, ME1-cKO and ME2-cKO). 

Id1-cKO and ME-cKOs showed a slight drop of left ventricular ejection fraction (LVEF) under HFD conditions compared to WT controls (loxP- cre+ and loxP+ cre-). However, the transcriptome showed a distinct upregulation of a specific gene pattern in WT animals (n=6, DESeq2 FDR<0.05), that was missing in ID1-cKO animals. These genes (e.g., Aldh1l2 fc WT 1.47 vs. fc KO 0.87, Pdk1 fc WT 1.21 vs. fc KO 0.78, Acot3 fc WT 2.31 vs. fc KO 1.58Cpt1b fc WT 1.28 vs. fc KO 1.02 and Gpam fc WT 1.52 vs. fc KO 0.75) are known to regulate fatty acid metabolism (FAM). Strikingly, single deletion of one H3K4me1-positive enhancer with maintained expression of the gene Id1 impaired the transcriptional response to the same extent. This Id1- independent regulation is indicating an enhancer-dependent mechanism. We performed a pulldown of nuclear proteins, bound to biotin-labeled ME2 in vitro to discover upstream regulating proteins. From 514 uniquely bound epigenetic modifiers (e.g., EHMT1, EHMT2), we found 21 transcription factors. We performed a complementary DeNovo transcription factor analysis of the promoter regions of the enhancer-dependent FAM-genes. By overlapping mass spectrometry data with the in silico analysis, we identified the retinoid acid receptor (ROR) binding motif (p=1e-13) and the related receptors (RxRx) as the regulatory element of the SE. Reporter assays showed activation of the FAM promoters and the ME2 in response to RxR overexpression and exposure to all-trans retinoid acid (ATRA). Functionally, the PDK1/4-phosphorylation target pyruvate dehydrogenase (PDH) was hyperphosphorylated at Serin 293 in WT animals, but not in ME2-cKO (PDH pS293/PDH: FC WT HFD/LFD: 6.46; FC ME2 HFD/LFD: 1.84, p=0.002, Mann-Whitney Test), indicating a missing metabolic flexibility upon SE deletion.

In human myocardial samples from obese donors (n= 6, mean BMI: 36.6 kg/m± 1.7 SEM), Id1, ME1 and ME2 were hyperacetylated at H3K27 compared to samples from normal weighted donors (n=4, mean BMI: 21.2 kg/m± 1.1 SEM). In conclusion, we discovered an evolutionary conserved mechanism of diet-induced retinoid acid-dependent epigenetic imprinting regulating adaptive FAM and glycolysis in the heart. 

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