https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Universitätsklinikum Heidelberg Institut für experimentelle Kardiologie Heidelberg, Deutschland; 3Deutsches Krebsforschungszentrum (DKFZ) Heidelberg, Deutschland; 4Universitätsklinikum Würzburg Deutsches Zentrum für Herzinsuffizienz/DZHI Würzburg, Deutschland
The response to environmental stress may be stored in epigenetic modifications. We hypothesized that regulation of cardiac enhancers might be an early event under metabolic stress which alters subsequent stress response.
C57BL/6J mice were subjected to either Low Fat Diet (LFD) or High Fat Diet (HFD). We included animals on transient HFD that completely recovered from the diet. On the epigenetic level, we found 21.934 H3K4me1-positive enhancers and 330 differentially regulated H3K27ac-positive enhancers after HFD with a false discovery rate of FDR<0.1. Despite a high reversibility of the HFD-induced enhancer one superenhancer (SE) was persistently activated. In human myocardial samples from obese donors (n= 6, mean BMI: 36.6 kg/m2 ± 1.7 SEM), the same SE was hyperacetylated at H3K27 compared to samples from normal weighted donors (n=4, mean BMI: 21.2 kg/m2 ± 1.1 SEM).
To investigate the biological consequences of the metabolic SE, we generated cardiomyocyte-specific metabolic enhancer knockout (cME-KO) lines of different parts of the SE using CRISPR/Cas9.
Especially, cME2-KO mice showed a reduced activation of fatty acid metabolism (FAM) genes after HFD (e.g., Pdk1 fc WT 1.21 vs. ME2-cKO 1.05, Acot3 fc WT 2.39 ME2-cKO 2.19, Cpt1b fc WT 1.28 vs. ME2-cKO 1,09 and Gpam fc WT 1.52 vs. ME2-cKO 1.29).Pyruvate dehydrogenase (PDH), a known phospho-target of Pdks, 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. This correlated with a more reduced status in NADH/FAD ratios and higher reactive oxygen species levels in ME2-cKO animals under HFD.
In De Novo transcription factor analysis and in a complementary approach of a biotin-labeled ME2 pulldown followed by mass spectrometry, we deciphered a prominent role of retinoid acid receptors (RxRs) for the enhancer-dependent gene regulation. Overexpression of enhancer-RNA (eRNA) and RxRa-GFP in-vitro showed increased formation of DNA condensates, that are attributed to long-range interactions. Consequently, HiC-seq experiments showed attenuated intrachromosomal interactions on DNA level in ME2-cKO under HFD of the identified SE, predominantly at RxR binding sites, including the promoter region of Pdk1.
To assess the impact of genetic ME2 deletion on the response to heart failure, we applied overload by O-ring aortic banding (ORAB) in ME2-cKO and WT animals after a history of obesity. We observed aggravated heart failure (mean LVEF, WT sham: 71.2%; WT ORAB: 54.1%; ME2-cKO ORAB: 35.1%, p=0.03, Mann-Whitney Test). Transcriptional analysis showed an additional downregulation of the RxR target genes (Pdk1, Acot3, Cpt1b and Gpam) in heart failure in ME2-cKO mice. Accordingly, genes involved in pathological cardiac remodeling, including Co1a1, Col3a1, Nppb and Myh7 were highly upregulated in ME2-cKO.
In conclusion, we discovered an evolutionary conserved mechanism of diet-induced retinoid acid-dependent epigenetic imprinting that modulates cardiac response to heart failure.